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Standardized naming systems are essential to integrate and unify distinct research fields, and to link multi-species data within and across kingdoms. We conducted a comprehensive survey of cytoplasmic ribosomal proteins (CRPs) in the dicot model Arabidopsis thaliana and the monocot model rice, noting that the standardized naming system has not been widely adopted in the plant community. We generated a database linking the old classical names to their updated and compliant names. We also explored the sequences, molecular evolution, and structural and functional characteristics of all plant CRP families, emphasizing evolutionarily conserved and plant-specific features through cross-kingdom comparisons. Unlike fungal CRP paralogs that were mainly created by whole-genome duplication (WGD) or retroposition under a concerted evolution mode, plant CRP genes evolved primarily through both WGD and tandem duplications in a rapid birth-and-death process. We also provide a web-based resource (http://www.plantcrp.cn/) with the aim of sharing the latest knowledge on plant CRPs and facilitating the continued development of a standardized framework across the entire community.

Small RNA (sRNA) population in plants comprises of primarily micro RNAs (miRNAs) and small interfering RNAs (siRNAs). MiRNAs play important roles in plant growth and development. The miRNA-derived secondary siRNAs are usually known as phased siRNAs, including phasiRNAs and tasiRNAs. The miRNA and phased siRNA biogenesis mechanisms are highly conserved in plants. However, their functional conservation and diversification may differ in maize. In the past two decades, lots of miRNAs and phased siRNAs have been functionally identified for curbing important maize agronomic traits, such as those related to developmental timing, plant architecture, sex determination, reproductive development, leaf morphogenesis, root development and nutrition, kernel development and tolerance to abiotic stresses. In contrast to Arabidopsis and rice, studies on maize miRNA and phased siRNA biogenesis and functions are limited, which restricts the small RNA-based fundamental and applied studies in maize. This review updates the current status of maize miRNA and phased siRNA mechanisms and provides a survey of our knowledge on miRNA and phased siRNA functions in controlling agronomic traits. Furthermore, improvement of those traits through manipulating the expression of sRNAs or their targets is discussed.

MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development and abiotic stresses. To date, studies have mainly focused on the roles of individual miRNAs, however, a few have addressed the interactions among multiple miRNAs. In this study, we investigated the interplay and regulatory circuit between miR160 and miR165/166 and its effect on leaf development and drought tolerance in Arabidopsis using Short Tandem Target Mimic (STTM). By crossing STTM160 Arabidopsis with STTM165/166, we successfully generated a double mutant of miR160 and miR165/166. The double mutant plants exhibited a series of compromised phenotypes in leaf development and drought tolerance in comparison to phenotypic alterations in the single STTM lines. RNA-seq and qRT-PCR analyses suggested that the expression levels of auxin and ABA signaling genes in the STTM-directed double mutant were compromised compared to the two single mutants. Our results also suggested that miR160-directed regulation of auxin response factors (ARFs) contribute to leaf development via auxin signaling genes, whereas miR165/166- mediated HD-ZIP IIIs regulation confers drought tolerance through ABA signaling. Our studies further indicated that ARFs and HD-ZIP IIIs may play opposite roles in the regulation of leaf development and drought tolerance that can be further applied to other crops for agronomic traits improvement.

High-throughput sequencing was employed to investigate the expression of miRNAs and their target genes in superior and inferior seeds of Aikang 58. Small RNA sequencing revealed 620 conserved and 64 novel miRNAs in superior grains, and 623 conserved and 66 novel miRNAs in inferior grains. Among these, 97 known miRNAs, and eight novel miRNAs showed differential expression between the superior and inferior seeds. Degradome sequencing revealed at least 140 candidate target genes associated with 35 miRNA families during the development of superior and inferior seeds. GO and KEGG pathway analysis showed that the differentially expressed miRNAs, both conserved and novel, were likely involved in hormone production, carbohydrate metabolic pathways, and cell division. We validated eight known and four novel grain development-related miRNAs and their target genes by quantitative real-time polymerase chain reaction to ensure the reliability of small RNA and degradome-seq results. Of these, miR160 and miR165/166 were knocked down in Arabidopsis using short-tandem target mimic (STTM160 and STTM165/166) technology, which confirmed their roles in seed development. Specifically, STTM160 showed significantly smaller grain size, lower grain weight, shorter siliques length, shorter plant height, and more serrated leaves, whereas STTM165/166 showed decreased seed number, disabled siliques, and curled upward leaves.

Background Photoperiod-sensitivity is a critical endogenous regulatory mechanism for plant growth and development under specific environmental conditions, while phosphate and sucrose signaling processes play key roles in cell growth and organ initiation. MicroRNA399 is phosphate-responsive, but, whether it has roles in other metabolic processes remains unknown. Results MicroRNA399 was determined to be sucrose-responsive through a microRNA array assay. High levels of sucrose inhibited the accumulation of microRNA399 family under phosphate starvation conditions in Arabidopsis thaliana. Similarly, exogenous sucrose supplementation also reduced microRNA399 expression in maize at developmental transition stages. RNA sequencing of a near-isogenic line(photoperiod-sensitive) line and its recurrent parent Huangzao4, a photoperiod-insensitive line, was conducted at various developmental stages. Members of microRNA399 family were down-regulated under long-day conditions in the photoperiod-sensitive near-isogenic line that accumulated more sucrose in vivo compared with the control line Huangzao4. Conclusion MicroRNA399s may play central roles in the integration of sucrose sensing and photoperiodic responses under long day conditions in maize. Electronic supplementary material The online version of this article (10.1186/s12870-018-1460-9) contains supplementary material, which is available to authorized users.

MicroRNA 165 and 166 (miR165/166) is composed of nine members and targets five members (PHB, PHV, REV, ATHB8 and ATHB15) of the HD-ZIP III transcription factor family. Mutants generated by traditional methods could hardly reveal the overall functions of miR165/166 in plant development. In this study, the expressions of all miR165/166 members were simultaneously blocked by over-expressing STTM165/166-31 in Arabidopsis and tomato for functional dissection of miR165/166 family. Following a down-regulation of over 90% endogenous miR165/166, the target HD-ZIP III genes were correspondingly up-regulated in the STTM transgenic Arabidopsis and tomato plants. Notably, the STTM165/166-31 over-expressed Arabidopsis and tomato displayed pleiotropic effects on development which were not frequently observed in previously identified genetic mutants of either individual miR165/166 gene or any of the five target genes. Furthermore, the transgenic Arabidopsis showed increased IAA content and decreased IAA sensitivity accompanied by enhanced expressions of genes responsible for auxin biosynthesis and signaling, suggesting possible roles of auxin in mediation of miR165/166-regulated processes. Importantly, the transgenic Arabidopsis exhibited the improved behavior under salt stress. Overall, such diverse variations in plant development and physiological process revealed by STTM165/166 demonstrate a key role of miR165/166-mediated network in regulating plant development and responses to abiotic stresses.

microRNAs (miRNAs) are endogenous small non-coding RNAs that bind to mRNAs and target them for cleavage and/or translational repression, leading to gene silencing. We previously developed short tandem target mimic (STTM) technology to deactivate endogenous miRNAs in Arabidopsis. Here, we created hundreds of STTMs that target both conserved and species-specific miRNAs in Arabidopsis, tomato, rice, and maize, providing a resource for the functional interrogation of miRNAs. We not only revealed the functions of several miRNAs in plant development, but also demonstrated that tissue-specific inactivation of a few miRNAs in rice leads to an increase in grain size without adversely affecting overall plant growth and development. RNA-seq and small RNA-seq analyses of STTM156/157 and STTM165/166 transgenic plants revealed the roles of these miRNAs in plant hormone biosynthesis and activation, secondary metabolism, and ion-channel activity-associated electrophysiology, demonstrating that STTM technology is an effective approach for studying miRNA functions. To facilitate the study and application of STTM transgenic plants and to provide a useful platform for storing and sharing of information about miRNA-regulated gene networks, we have established an online Genome Browser (https://blossom.ffr.mtu.edu/designindex2.php) to display the transcriptomic and miRNAomic changes in STTM-induced miRNA knockdown plants.

The plant hormones brassinosteroids (BRs) participate in light-mediated regulation of plant growth, although the underlying mechanisms are far from being fully understood. In addition, the function of the core transcription factor in the BR signaling pathway, BRI1-EMS-SUPPRESSOR 1 (BES1), largely depends on its phosphorylation status and its protein stability, but the regulation of BES1 is not well understood. Here, we report that SINA of Arabidopsis thaliana (SINATs) specifically interact with dephosphorylated BES1 and mediate its ubiquitination and degradation. Our genetic data demonstrated that SINATs inhibit BR signaling in a BES1-dependent manner. Interestingly, we found that the protein levels of SINATs were decreased in the dark and increased in the light, which changed BES1 protein levels accordingly. Thus, our study not only uncovered a new mechanism of BES1 degradation but also provides significant insights into how light conditionally regulates plant growth through controlling accumulation of different forms of BES1.

Arabidopsis glycogen synthase kinase 3 (GSK3)-like kinases have versatile functions in plant development and in responding to abiotic stresses. Although physiological evidence suggested a potential role of GSK3-like kinases in abscisic acid (ABA) signaling, the underlying molecular mechanism was largely unknown. Here we identified members of Snf1-related kinase 2s (SnRK2s), SnRK2.2 and SnRK2.3, that can interact with and be phosphorylated by a GSK3-like kinase, brassinosteroid insensitive 2 (BIN2). bin2-3 bil1 bil2, a loss-of-function mutant of BIN2 and its two closest homologs, BIN2 like 1 (BIL1) and BIN2 like 2 (BIL2), was hyposensitive to ABA in primary root inhibition, ABA-responsive gene expression, and phosphorylating ABA Response Element Binding Factor (ABF) 2 fragment by in-gel kinase assays, whereas bin2-1, a gain-of-function mutation of BIN2, was hypersensitive to ABA, suggesting that these GSK3-like kinases function as positive regulators in ABA signaling. Furthermore, BIN2 phosphorylated SnRK2.3 on T180, and SnRK2.3(T180A) had decreased kinase activity in both autophosphorylation and phosphorylating ABFs. Bikinin, a GSK3 kinase inhibitor, inhibited the SnRK2.3 kinase activity and its T180 phosphorylation in vivo. Our genetic analysis further demonstrated that BIN2 regulates ABA signaling downstream of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS receptors and clade A protein phosphatase 2C but relies on SnRK2.2 and SnRK2.3. These findings provide significant insight into the modulation of ABA signaling by Arabidopsis GSK3-like kinases.

In plants, microRNAs (miRNAs) regulate more than hundred target genes comprising largely transcription factors that control growth and development as well as stress responses. However, the exact functions of miRNA families could not be deciphered because each miRNA family has multiple loci in the genome, thus are functionally redundant. Therefore, an ideal approach to study the function of a miRNA family is to silence the expression of all members simultaneously, which is a daunting task. However, this can be partly overcome by Target Mimic (TM) approach that can knockdown an entire miRNA family. STTM is a modification of TM approach and complements it. STTMs have been successfully used in monocots and dicots to block miRNA functions. miR159 has been shown to be differentially regulated by various abiotic stresses including ABA in various plant species. Here, we describe in detail the protocol for designing STTM construct to block miR159 functions in Arabidopsis, with the potential to apply this technique on a number of other stress-regulated miRNAs in plants.

RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5’ products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC. DOI: http://dx.doi.org/10.7554/eLife.24466.001, eLife digest DNA contains all the information needed to build a body, yet molecules of RNA carry these instructions to the sites in the cell where they can be used. Cells must control how much RNA they produce in order to ensure that they develop properly and can respond well to their environment. RNA silencing refers to a collection of mechanisms that use smaller RNA molecules called microRNAs to incapacitate certain RNA molecules and selectively switch off the genes that encode them to stop more from being made. One key player in RNA silencing is the multi-protein complex called RISC, which contains microRNA and a group of proteins called AGOs. Once the microRNA has identified its RNA target, the AGOs cut the RNA into two pieces, known as the 5’ cleavage fragment and 3’ cleavage fragment. The two resulting fragments need to be cleared away swiftly, so that the RISC can move on to the next target. While it was known how the 3’ cleavage fragment was removed, it was less clear how the 5’ cleavage fragment was dealt with. Previous studies had shown that the 5’ cleavage fragment was marked with a chemical called uridine, which somehow signals to the RISC that this fragment needs to be destroyed. Now, using biochemical techniques, Zhang et al. have identified two new proteins in the model plant Arabidopsis that attach to the AGO proteins and degrade the 5’ cleavage fragments that are marked with uridine. The two proteins are named RICE1 and RICE2. Zhang et al. then analyzed the three-dimensional shape of RICE1 and identified the ‘active’ region that is responsible for degrading the RNA fragments. When these active regions were blocked, the microRNA levels were low, but the uridine-marked 5’ cleavage fragments were high. Also, the RISC complex could not work properly, which lead to problems during the development of the plant. These results suggest that RICE proteins degrade 5’ cleavage fragments modified with uridine to activate RISC. RICE proteins are conserved between plants and animals, and it is likely that their counterparts in humans will have a similar role to the plant proteins. The next challenge will be to explore how RICE proteins work in more details, which may lead to new ways to manipulate the levels of microRNAs to change the architecture of the plant and to improve their tolerance to various stress conditions. DOI: http://dx.doi.org/10.7554/eLife.24466.002

The function of miR165/166 in plant growth and development has been extensively studied, however, its roles in abiotic stress responses remain largely unknown. Here, we report that reduction in the expression of miR165/166 conferred a drought and cold resistance phenotype and hypersensitivity to ABA during seed germination and post-germination seedling development. We further show that the ABA hypersensitive phenotype is associated with a changed transcript abundance of ABA-responsive genes and a higher expression level of ABI4, which can be directly regulated by a miR165/166 target. Additionally, we found that reduction in miR165/166 expression leads to elevated ABA levels, which occurs at least partially through the increased expression of BG1, a gene that is directly regulated by a miR165/166 target. Taken together, our results uncover a novel role for miR165/166 in the regulation of ABA and abiotic stress responses and control of ABA homeostasis., Author Summary Functions of miRNAs in plant development and stress responses have been extensively studied. However, little is known about how a miRNA may perform critical functions in both plant development and abiotic stress responses. One well-known miRNA, miR165/166, has critical roles in plant development. In this study, we show that this miRNA also has important functions in ABA and abiotic stress responses. Since the expression level of miR165/166 can be reduced to different extents using short tandem target mimicry (STTM), in the present work, we used STTM165/166 transformants with moderate developmental phenotype to examine its potential role in abiotic stress responses. Our results show that miR165/166 plays critical roles in drought and cold stress resistance as well as in ABA responses. Our work reveals that miR165/166-mediated regulatory module is linked with ABA responses and homeostasis through ABI4 and BG1.

Sucrose induces the expression of miR398 and the production of miR398 is controlled by SQUAMOSA promoter binding protein-like7 (SPL7) transcription factor under copper deficiency in Arabidopsis thaliana. Little else, however, is known about the sucrose-regulated copper homeostasis in Arabidopsis. Here, we employed the microarray technology to screen for sucrose-responsive microRNAs (miRNAs), and identified several candidates using northern blotting. In addition to miR398, miR408, miR319, and miR160 are also responsive to sucrose. Furthermore, the induction of miR398 and miR408 by sucrose depends on the presence of SPL7. MiR398 and miR408 induction by high sucrose was eliminated by high copper. Meanwhile, high sucrose treatment strongly suppressed copper accumulation. Knock-down of SPL7 dramatically reduced copper accumulation in plants under low sucrose, but this effect was compromised in the presence of high levels of sucrose. Taken together, these results suggest a connection between sucrose signaling and copper accumulation in cells, and that sucrose-regulated copper accumulation is both SPL7-dependent and SPL7-independent in Arabidopsis.

MicroRNAs (miRNAs) and other endogenous small RNAs act as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. The interrogation of small RNA functions requires an effective, widely applicable method to specifically block small RNA function. Here, we report the development of a highly effective technology that targets specific endogenous miRNAs or small interfering RNAs for destruction in Arabidopsis thaliana. We show that the expression of a short tandem target mimic (STTM), which is composed of two short sequences mimicking small RNA target sites, separated by a linker of an empirically determined optimal size, leads to the degradation of targeted small RNAs by small RNA degrading nucleases. The efficacy of the technology was demonstrated by the strong and specific developmental defects triggered by STTMs targeting three miRNAs and an endogenous siRNA. In summary, we developed an effective approach for the destruction of endogenous small RNAs, thereby providing a powerful tool for functional genomics of small RNA molecules in plants and potentially animals.

MicroRNAs (miRNAs) are endogenous small RNAs of ~22 nucleotides (nt) that play a key role in down regulation of gene expression at the post-transcriptional level in plants and animals. Various studies have identified numerous miRNAs that were either up regulated or down regulated upon stress treatment. Here, we sought to understand the temporal regulation of miRNAs in different plant species under abscisic acid (ABA) and salt (NaCl) stress. Our results showed that the regulation of miR398 in response to ABA and salt stress was more dynamic in plants than previously reported. In poplars, miR398 was first induced upon 3-4 h of ABA or salt stress. However, this induction declined after 48 h and finally accumulated again over a prolonged stress (72 h). We referred to this kind of regulation as dynamic regulation. In contrast, such dynamic regulation of miR398 under salt stress was completely absent in Arabidopsis, in which miR398 was steadily and unidirectionally suppressed. Interestingly, ABA treatment caused a deviate dynamic regulation of miR398 in Arabidopsis, showing an opposite response as compared to that in poplars. We referred to the difference in regulation between Arabidopsis and poplars as differential regulation. Furthermore, the expression of the miR398 target, copper superoxide dismutase1 (CSD1), was in reverse correlation with the miR398 level, suggesting a control of this specific target expression predominantly by miR398 under abiotic stress. Together, these data consistently show a correlated regulation between miR398 and its representative target, CSD1, by ABA and salt stresses, and raise the possibility that regulation of miRNAs in plants is twofold: a dynamic regulation within a plant species and a differential regulation between different plant species.

Lysine catabolism in plants is initiated by a bifunctional LKR/SDH (lysine-ketoglutarate reductase/saccharopine dehydrogenase) enzyme encoded by a single LKR/SDH gene. Yet, the AtLKR/SDH gene of Arabidopsis also encodes a second gene product, namely a monofunctional SDH. To elucidate the regulation of lysine catabolism in Arabidopsis through these two gene products of the AtLKR/SDH gene, an analysis was carried out on the effects of the hormones, abscisic acid and jasmonate, as well as various metabolic and stress signals, including lysine itself, on their mRNA and protein levels. The response of the two gene products to the various treatments was only partially co-ordinated, but the levels of the monofunctional SDH mRNA and protein were always in excess over their bifunctional LKR/SDH counterparts. These results suggest that lysine catabolism is regulated primarily by the first enzyme LKR, while the excess level of SDH enables efficient flux of lysine catabolism following the LKR step. Analysis of transgenic plants expressing beta-glucoronidase fusion constructs with the AtLKR/SDH and monofunctional AtSDH promoters demonstrated that transcriptional regulation contributes to the modulation of expression of the bifunctional LKR/SDH and monofunctional SDH gene products in response to hormonal and metabolic signals. To test whether the enhanced expression of the LKR/SDH gene under various hormonal and metabolic signals is correlated with enhanced lysine catabolism, wild-type Arabidopsis and a knockout mutant lacking lysine catabolism were exposed to abscisic acid and sugar starvation. Free lysine accumulated to significantly higher levels in this knockout mutant than in the wild-type plants.

Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in animals and plants. To elucidate the biochemical signification of the linkage between the two enzymes of LKR/SDH, namely lysine ketoglutarate and saccharopine dehydrogenase, we employed various truncated and mutated Arabidopsis LKR/SDH polypeptides expressed in yeast. Activity analyses of the different recombinant polypeptides under conditions of varying NaCl levels implied that LKR, but not SDH activity, is regulated by functional interaction between the LKR and SDH domains, which is mediated by the structural conformation of the linker region connecting them. Because LKR activity of plant LKR/SDH enzymes is also regulated by casein kinase 2 phosphorylation, we searched for such potential regulatory phosphorylation sites using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and site-directed mutagenesis. This analysis identified Ser-458 as a candidate for this function. We also tested a hypothesis suggesting that an EF-hand-like sequence at the C-terminal part of the LKR domain functions in a calcium-dependent assembly of LKR/SDH into a homodimer. We found that this region is essential for LKR activity but that it does not control a calcium-dependent assembly of LKR/SDH. The relevance of our results to the in vivo function of LKR/SDH in lysine catabolism in plants is discussed. In addition, because the linker region between LKR and SDH exists only in plants but not in animal LKR/SDH enzymes, our results suggest that the regulatory properties of LKR/SDH and, hence, the regulation of lysine catabolism are different between plants and animals.

Both plants and animals catabolize lysine (Lys) via two consecutive enzymes, Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide encoded by a single LKR/SDHgene. We have previously shown that the ArabidopsisLKR/SDH gene also encodes a monofunctional SDH that is transcribed from an internal promoter. In the present report, we have identified two cDNAs derived from cotton (Gossypium hirsutum) boll abscission zone that encode a novel enzymatic form of Lys catabolism, i.e. a catabolic monofunctional LKR. The monofunctional LKR mRNA is also encoded by theLKR/SDH gene, using two weak polyadenylation sites located within an intron. In situ mRNA hybridization and quantitative reverse transcriptase-polymerase chain reaction analyses also suggest that the cotton monofunctional LKR is relatively abundantly expressed in parenchyma cells of the abscission zone. DNA sequence analysis of theLKR/SDH genes of Arabidopsis, maize (Zea mays), and tomato (Lycopersicon esculentum) suggests that these genes can also encode a monofunctional LKR mRNA by a similar mechanism. To test whether the LKR/SDH and monofunctional LKR enzymes possess different biochemical properties, we used recombinant Arabidopsis LKR/SDH and monofunctional LKR enzymes expressed in yeast (Saccharomyces cerevisiae) cells. The K m of the monofunctional LKR to Lys was nearly 10-fold lower than its counterpart that is linked to SDH. Taken together, our results suggest that theLKR/SDH locus of plants is a super-composite locus that can encode three related but distinct enzymes of Lys catabolism. These three enzymes apparently operate in concert to finely regulate Lys catabolism during plant development.

Summary Both plants and animals catabolise lysine via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide. We recently demonstrated that Arabidopsis plants possess not only a bifunctional LKR/SDH but in addition a monofunctional SDH enzyme. We also speculated that these two enzymes may be controlled by a single gene (G. Tang et al. Plant Cell, 1997, 9, 1305–1316). By expressing several epitope-tagged and GUS reporter constructs, we demonstrate in the present study that the Arabidopsis monofunctional SDH is encoded by a distinct gene, which is, however, nested entirely within the coding and 3′ non-coding regions of the larger bifunctional LKR/SDH gene. The entire open reading frame of the monofunctional SDH gene, as well as some components of its promoter, are also parts of the translated coding sequence of the bifunctional LKR/SDH gene. These special structural characteristics, combined with the fact that the two genes encode simultaneously two metabolically related but distinct enzymes, render the LKR/SDH locus a novel type of a composite locus. Not all plant species possess an active monofunctional SDH gene and the production of this enzyme is correlated with an increased flux of lysine catabolism. Taken together, our results suggest that the composite LKR/SDH locus serves to control an efficient, highly regulated flux of lysine catabolism

As in many bacterial species, the first enzymatic reaction of the aspartate-family pathway in plants is mediated by several isozymes of aspartate kinase (AK) that are subject to feedback inhibition by the end-product amino acids lysine or threonine. So far, only cDNAs and genes encoding threonine-sensitive AKs have been cloned from plants. These were all shown to encode polypeptides containing two linked activities, namely AK and homoserine dehydrogenase (HSD), similar to the Escherichia coli thrA gene encoding a threonine-sensitive bifunctional AK/HSD isozyme. In the present report, we describe the cloning of a new Arabidopsis thaliana cDNA that is relatively highly homologous to the E. coli lysC gene encoding the lysine-sensitive AK isozyme. Moreover, similar to the bacterial lysine-sensitive AK, the polypeptide encoded by the present cDNA is monofunctional and does not contain an HSD domain. These observations imply that our cloned cDNA encodes a lysine-sensitive AK. Southern blot hybridization detected a single gene highly homologous to the present cDNA, plus an additional much less homologous gene. This was confirmed by the independent cloning of an additional Arabidopsis cDNA encoding a lysine-sensitive AK (see accompanying paper). Northern blot analysis suggested that the gene encoding this monofunctional AK cDNA is abundantly expressed in most if not all tissues of Arabidopsis.

The utility of artificial microRNAs (amiRNAs) to induce loss of gene function has been reported for many plant species, but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable. In this study, expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation. A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments. Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs. This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants.

Ozone is a model abiotic elicitor of reactive oxygen species (ROS). ROS are important oxidative signaling molecules coordinating plant development and responses to biotic and abiotic stresses. Recently, microRNAs have been described as important players in regulating stress responses in plants. In this research we examined the miRNAs that are differentially expressed early in response to ozone in the Arabidopsis thaliana ecotype Col-0 that is tolerant to this oxidant. We used a plant miRNA array to identify 22 miRNA families that are differentially expressed within one hour of ozone fumigation. Majority of these miRNAs were also reported in response to UV-B stress. Analysis of the miRNA target genes showed a strong negative correlation to the miRNA expression. In silico promoter analysis of miRNA genes identified several stress responsive cis-elements that were enriched in the promoters of ozone responsive genes. Majority of the target genes of ozone responsive miRNAs were associated with developmental processes. Based on these results we suggest that post-transcriptional gene regulation via miRNAs may aid in resource allocation by downregulating developmental processes to cater to the oxidative stress demands on plants.

Stem cells are crucial in morphogenesis in plants and animals. Much is known about the mechanisms that maintain stem cell fates or trigger their terminal differentiation. However, little is known about how developmental time impacts stem cell fates. Using Arabidopsis floral stem cells as a model, we show that stem cells can undergo precise temporal regulation governed by mechanisms that are distinct from, but integrated with, those that specify cell fates. We show that two microRNAs, miR172 and miR165/166, through targeting APETALA2 and type III homeodomain-leucine zipper (HD-Zip) genes, respectively, regulate the temporal program of floral stem cells. In particular, we reveal a role of the type III HD-Zip genes, previously known to specify lateral organ polarity, in stem cell termination. Both reduction in HD-Zip expression by over-expression of miR165/166 and mis-expression of HD-Zip genes by rendering them resistant to miR165/166 lead to prolonged floral stem cell activity, indicating that the expression of HD-Zip genes needs to be precisely controlled to achieve floral stem cell termination. We also show that both the ubiquitously expressed ARGONAUTE1 (AGO1) gene and its homolog AGO10, which exhibits highly restricted spatial expression patterns, are required to maintain the correct temporal program of floral stem cells. We provide evidence that AGO10, like AGO1, associates with miR172 and miR165/166 in vivo and exhibits “slicer” activity in vitro. Despite the common biological functions and similar biochemical activities, AGO1 and AGO10 exert different effects on miR165/166 in vivo. This work establishes a network of microRNAs and transcription factors governing the temporal program of floral stem cells and sheds light on the relationships among different AGO genes, which tend to exist in gene families in multicellular organisms., Author Summary Stem cells have the capacity to self renew while producing daughter cells that undergo differentiation. While some stem cells remain as stem cells throughout the life of an organism, others are programmed to terminate within developmental contexts. It is presumed that stem cell termination is simply the differentiation of stem cells into a specific cell type(s). Using floral stem cells as a model, we show that the temporally regulated termination of floral stem cells is genetically separable from stem cell differentiation, and thus we reveal the presence of a temporal program of stem cell regulation. We show that two microRNAs, miR172 and miR165/166, and two argonaute family proteins, ARGONAUTE1 (AGO1) and AGO10, regulate the termination of floral stem cells. We establish the homeodomain-leucine zipper (DH-Zip) genes, targets of miR165/166, as crucial factors in floral stem cell termination. While AGO1 is the major miRNA effector, the molecular function of AGO10 has been elusive. Here we demonstrate that AGO10 is also a miRNA effector in that AGO10 is associated with miRNAs in vivo and exhibits “slicer” activity in vitro. Despite the similar biochemical activities, AGO1 and AGO10 promote floral stem cell termination by exerting opposite effects on miR165/166.

MicroRNAs (miRNAs) are approximately 22-nucleotide (nt)-long non-coding RNAs that play a key role in plant development and abiotic stresses. We have developed a simple but effective array platform for profiling plant miRNAs from various plant species. The array is composed of 188 non-redundant miRNA probes that can detect both conserved and species-specific miRNAs from most plant species, including Arabidopsis, rice, and poplar. In this chapter, we describe a protocol for developing the miRNA array platform, which can be used to identify stress-responsive miRNAs in diverse plant species. Profiling of miRNAs in tobacco seedlings exposed to different abiotic stress conditions has revealed that a number of miRNAs, miR398, miR399, miR408, miR156, miR164, and miR168, were responsive to stresses. This comprehensive and easy-to-follow protocol will be useful for studying roles of miRNAs in plant stress response.

microRNAs (miRNAs) are a novel class of 21–24 nucleotide, endogenous, noncoding small ribonucleic acids (RNAs) that play critical roles in diverse biological processes such as: development, cellular differentiation, cell-cycle control, apoptosis and oncogenesis. In plants, roles of miRNAs in development have been extensively investigated in recent years. This review highlights and discusses the current knowledge about the roles of microRNAs in leaf development, patterning and polarity, floral identity and flower development, developmental phase transition and flowering time, shoot and root development, vascular development and hormone signalling for plant development. Keywords: microRNAs; plant development; gene regulation; transcription factors; arabidopsis

Although RNA silencing was first discovered in plants, thus far it has been studied biochemically only in animals, where it is known as RNA interference (RNAi). In animals, two components of the RNAi pathway have been identified: Dicer, a multidomain RNase III that converts long double-stranded RNA (dsRNA) into small interfering RNA (siRNA) and the RNA-induced silencing complex (RISC), as siRNA-containing protein-RNA complex that targets complementary mRNA for destruction. We have developed methods for the biochemical dissection of plant RNA silencing. In this chapter, we describe in detail how to use wheat germ extract to study two distinct Dicer-like activities, RNA-dependent RNA polymerase (RdRP), and endogenous microRNA-programmed RISC activities. These comprehensive protocols should prove useful in the further dissection of the plant RNA silencing pathway, as well as for the validation of the predicted targets of endogenous plant microRNAs.

MicroRNAs (miRNAs) are approximately 22-nucleotide noncoding RNAs that can regulate gene expression by directing mRNA degradation or inhibiting productive translation. Dominant mutations in PHABULOSA (PHB) and PHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA-guided cleavage of these mRNAs in vitro. Here, we confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb-d mutants. In planta, disrupting miRNA pairing near the center of the miRNA complementary site had far milder developmental consequences than more distal mismatches. These differences correlated with differences in miRNA-directed cleavage efficiency in vitro, where mismatch scanning revealed more tolerance for mismatches at the center and 3' end of the miRNA compared to mismatches to the miRNA 5' region. In this respect, miR165/166 resembles animal miRNAs in its pairing requirements. Pairing to the 5' portion of the small silencing RNA appears crucial regardless of the mode of post-transcriptional repression or whether it occurs in plants or animals, supporting a model in which this region of the silencing RNA nucleates pairing to its target.

RNA silencing phenomena were first discovered in plants, yet only the RNA interference pathway in animals has been subject to biochemical analysis. Here, we extend biochemical analysis to plant RNA silencing. We find that standard wheat germ extract contains Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs, as well as an RNA-dependent RNA polymerase activity that can convert exogenous single-stranded RNA into dsRNA. In this plant embryo extract, an endogenous microRNA (miRNA) that lacks perfect complementarity to its RNA targets nonetheless acts as a small interfering RNA. The miRNA guides an endonuclease to cleave efficiently wild-typeArabidopsis PHAVOLUTAmRNA, but not a dominant mutant previously shown to perturb leaf development. This finding supports the view that plant miRNAs direct RNAi and that miRNA-specified mRNA destruction is important for proper plant development. Thus, endonuclease complexes guided by small RNAs are a common feature of RNA silencing in both animals and plants.

Lysine is a nutritionally important essential amino acid whose level in plants is largely regulated by the rate of its synthesis. In some plant tissues and under some stress conditions, however, lysine is also efficiently catabolized into glutamate and several other stress-related metabolites by novel mechanisms of metabolic regulation. Lysine catabolism is important for mammalian brain function; it is possible that the generation of glutamate regulates nerve transmission signals via glutamate receptors. Plants also possess homologues of animal glutamate receptors. It is thus likely that lysine catabolism also regulates various plant processes via these receptors.

Plants possess both anabolic and catabolic pathways for the essential amino acid lysine (Lys). However, although the biosynthetic pathway was clearly shown to regulate Lys accumulation in plants, the functional significance of Lys catabolism has not been experimentally elucidated. To address this issue, we have isolated an Arabidopsis knockout mutant with a T-DNA inserted into exon 13 of the gene encoding Lys ketoglutarate reductase/saccharopine dehydrogenase. This bifunctional enzyme controls the first two steps of Lys catabolism. The phenotype of the LKR/SDH knockout was indistinguishable from wild-type plants under normal growth conditions, suggesting that Lys catabolism is not an essential pathway under standard growth conditions. However, mature seeds of the knockout mutant over-accumulated Lys compared with wild-type plants. This report provides the first direct evidence for the functional significance of Lys catabolism in regulating Lys accumulation in seeds. Such a knockout mutant may also provide new perspectives to improve the level of the essential amino acid Lys in plant seeds.

Whereas plants and animals use the α-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine–oxoglutarate reductase (lysine–ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the α-amino adipic acid pathway in plants.

In plant and mammalian cells, excess lysine is catabolized by a pathway that is initiated by two enzymes, namely, lysine-ketoglutarate reductase and saccharopine dehydrogenase. In this study, we report the cloning of an Arabidopsis cDNA encoding a bifunctional polypeptide that contains both of these enzyme activities linked to each other. RNA gel blot analysis identified two mRNA bands-a large mRNA containing both lysine-ketoglutarate reductase and saccharopine dehydrogenase sequences and a smaller mRNA containing only the saccharopine dehydrogenase sequence. However, DNA gel blot hybridization using either the lysine-ketoglutarate reductase or the saccharopine dehydrogenase cDNA sequence as a probe suggested that the two mRNA populations apparently are encoded by the same gene. To test whether these two mRNAs are functional, protein extracts from Arabidopsis cells were fractionated by anion exchange chromatography. This fractionation revealed two separate peaks-one containing both coeluted lysine-ketoglutarate reductase and saccharopine dehydrogenase activities and the second containing only saccharopine dehydrogenase activity. RNA gel blot analysis and in situ hybridization showed that the gene encoding lysine-ketoglutarate reductase and saccharopine dehydrogenase is significantly upregulated in floral organs and in embryonic tissues of developing seeds. Our results suggest that lysine catabolism is subject to complex developmental and physiological regulation, which may operate at gene expression as well as post-translational levels.