Knockdown of microRNA390 Enhances Maize Brace Root Growth.

Brace root architecture is a critical determinant of maize's stalk anchorage and nutrition uptake, influencing root lodging resistance, stress tolerance, and plant growth. To identify the key microRNAs (miRNAs) in control of maize brace root growth, we performed small RNA sequencing using brace root samples at emergence and growth stages. We focused on the genetic modulation of brace root development in maize through manipulation of miR390 and its downstream regulated auxin response factors (ARFs). In the present study, miR167, miR166, miR172, and miR390 were identified to be involved in maize brace root growth in inbred line B73. Utilizing short tandem target mimic (STTM) technology, we further developed maize lines with reduced miR390 expression and analyzed their root architecture compared to wild-type controls. Our findings show that STTM390 maize lines exhibit enhanced brace root length and increased whorl numbers. Gene expression analyses revealed that the suppression of miR390 leads to upregulation of its downstream regulated ARF genes, specifically ZmARF11 and ZmARF26, which may significantly alter root architecture. Additionally, loss-of-function mutants for ZmARF11 and ZmARF26 were characterized to further confirm the role of these genes in brace root growth. These results demonstrate that miR390, ZmARF11, and ZmARF26 play crucial roles in regulating maize brace root growth; the involved complicated molecular mechanisms need to be further explored. This study provides a genetic basis for breeding maize varieties with improved lodging resistance and adaptability to diverse agricultural environments.

Whole genome sequence analysis of shallot virus X from India reveals it to be a natural recombinant with positive selection pressure.

BACKGROUND: Shallots are infected by various viruses like Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and Shallot virus X (ShVX). In India, they have been found to be persistently infected by ShVX. ShVX also infects onion and garlic in combination with other carlaviruses and potyviruses. ShVX is a member of genus Allexivirus of family Alphaflexiviridae. ShVX has a monopartite genome, which is represented by positive sense single-stranded RNA. Globally, only six complete and 3 nearly complete genome sequences of ShV X are reported to date. This number is insufficient to measure a taxon's true molecular diversity. Moreover, the complete genome sequence of ShVX from Asia has not been reported as yet. Therefore, this study was undertaken to generate a complete genome sequence of ShVX from India. RESULTS: Shallot virus X (ShVX) is one of the significant threats to Allium crop production. In this study, we report the first complete genome sequence of the ShVX from India through Next-generation sequencing (NGS). The complete genome of the ShVX (Accession No. OK104171), from this study comprised 8911 nucleotides. In-silico analysis of the sequence revealed variability between this isolate and isolates from other countries. The dissimilarities are spread all over the genome specifically some non-coding intergenic regions. Statistical analysis of individual genes for site-specific selection indicates a positive selection in NABP region. The presence of a recombination event was detected in coat protein region. The sequence similarity percentage and phylogenetic analysis indicate ShVX Indian isolate is a distinctly different isolate. Recombination and site-specific selection may have a function in the evolution of this isolate. This is the first detailed study of the ShVX complete genome sequence from Southeast Asia. CONCLUSION: This study presents the first report of the entire genome sequence of an Indian isolate of ShVX along with an in-depth exploration of its evolutionary traits. The findings highlight the Indian variant as a naturally occurring recombinant, emphasizing the substantial role of recombination in the evolution of this viral species. This insight into the molecular diversity of strains within a specific geographical region holds immense significance for comprehending and forecasting potential epidemics. Consequently, the insights garnered from this research hold practical value for shaping ShVX management strategies and providing a foundation for forthcoming studies delving into its evolutionary trajectory.

A high-efficiency gene silencing in plants using two-hit asymmetrical artificial MicroRNAs.

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. They are produced through an enzyme-guided process called dicing and have an asymmetrical structure with two nucleotide overhangs at the 3' ends. Artificial microRNAs (amiRNAs or amiRs) are designed to mimic the structure of miRNAs and can be used to silence specific genes of interest. Traditionally, amiRNAs are designed based on an endogenous miRNA precursor with certain mismatches at specific positions to increase their efficiency. In this study, the authors modified the highly expressed miR168a in Arabidopsis thaliana by replacing the single miR168 stem-loop/duplex with tandem asymmetrical amiRNA duplexes that follow the statistical rules of miRNA secondary structures. These tandem amiRNA duplexes, called "two-hit" amiRNAs, were shown to have a higher efficiency in silencing GFP and endogenous PDS reporter genes compared to traditional "one-hit" amiRNAs. The authors also demonstrated the effectiveness of "two-hit" amiRNAs in silencing genes involved in miRNA, tasiRNA, and hormone signalling pathways, individually or in families. Importantly, "two-hit" amiRNAs were also able to over-express endogenous miRNAs for their functions. The authors compare "two-hit" amiRNA technology with CRISPR/Cas9 and provide a web-based amiRNA designer for easy design and wide application in plants and even animals.

The miR167-OsARF12 module regulates rice grain filling and grain size downstream of miR159.

Grain weight and quality are always determined by grain filling. Plant microRNAs have drawn attention as key targets for regulation of grain size and yield. However, the mechanisms that underlie grain size regulation remain largely unclear because of the complex networks that control this trait. Our earlier studies demonstrated that suppressed expression of miR167 (STTM/MIM167) substantially increased grain weight. In a field test, the yield increased up to 12.90%-21.94% because of a significantly enhanced grain filling rate. Here, biochemical and genetic analyses revealed the regulatory effects of miR159 on miR167 expression. Further analysis indicated that OsARF12 is the major mediator by which miR167 regulates rice grain filling. Overexpression of OsARF12 produced grain weight and grain filling phenotypes resembling those of STTM/MIM167 plants. Upon in-depth analysis, we found that OsARF12 activates OsCDKF;2 expression by directly binding to the TGTCGG motif in its promoter region. Flow cytometry analysis of young panicles from OsARF12-overexpressing plants and examination of cell number in cdkf;2 mutants verified that OsARF12 positively regulates grain filling and grain size by targeting OsCDKF;2. Moreover, RNA sequencing results suggested that the miR167-OsARF12 module is involved in the cell development process and hormone pathways. OsARF12-overexpressing plants and cdkf;2 mutants exhibited enhanced and reduced sensitivity to exogenous auxin and brassinosteroid (BR) treatment, confirming that targeting of OsCDKF;2 by OsARF12 mediates auxin and BR signaling. Our results reveal that the miR167-OsARF12 module works downstream of miR159 to regulate rice grain filling and grain size via OsCDKF;2 by controlling cell division and mediating auxin and BR signals.

CRISPR/Cas9 System: A Potential Tool for Genetic Improvement in Floricultural Crops.

Demand of flowers is increasing with time worldwide. Floriculture has become one of the most important commercial trades in agriculture. Although traditional breeding methods like hybridization and mutation breeding have contributed significantly to the development of important flower varieties, flower production and quality of flowers can be significantly improved by employing modern breeding approaches. Novel traits of significance have interest to consumers and producers, such as fragrance, new floral color, change in floral architecture and morphology, vase life, aroma, and resistance to biotic and abiotic stresses, have been introduced by genetic manipulation. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has recently emerged as a powerful genome-editing tool for accurately changing DNA sequences at specific locations. It provides excellent means of genetically improving floricultural crops. CRISPR/Cas system has been utilized in gene editing in horticultural cops. There are few reports on the utilization of the CRISPR/Cas9 system in flowers. The current review summarizes the research work done by employing the CRISPR/Cas9 system in floricultural crops including improvement in flowering traits such as color modification, prolonging the shelf life of flowers, flower initiation, and development, changes in color of ornamental foliage by genome editing. CRISPR/Cas9 gene editing could be useful in developing novel cultivars with higher fragrance and enhanced essential oil and many other useful traits. The present review also highlights the basic mechanism and key components involved in the CRISPR/Cas9 system.

MicroRNA Techniques: Valuable Tools for Agronomic Trait Analyses and Breeding in Rice.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the post-transcriptional level. Extensive studies have revealed that miRNAs have critical functions in plant growth, development, and stress responses and may provide valuable genetic resources for plant breeding research. We herein reviewed the development, mechanisms, and characteristics of miRNA techniques while highlighting widely used approaches, namely, the short tandem target mimic (STTM) approach. We described STTM-based advances in plant science, especially in the model crop rice, and introduced the CRISPR-based transgene-free crop breeding. Finally, we discussed the challenges and unique opportunities related to combining STTM and CRISPR technology for crop improvement and agriculture.

MicroRNAs meet with quantitative trait loci: Small powerful players in regulating quantitative yield traits in rice.

MicroRNAs (miRNAs) are small noncoding RNAs which regulate various functions related to growth, development, and stress responses in plants and animals. Rice, Oryza sativa, is one of the most important food crops of the world. In rice, a number of quantitative trait loci (QTL) controlling yield-related traits have been identified. Some of them are actually controlled by miRNAs, which control various yield-related quantitative traits in rice. On one hand, many of these miRNAs are found to regulate more than one yield-related traits, such as tillering, grain size, and branch number of a panicle. On the other hand, a rice yield-related trait is usually controlled by multiple miRNAs, for example, grain size being controlled by miR156, miR167, miR396, miR397, and miR1432. In rare case, a single miRNA may specifically regulate only one yield-related trait, such as, miR444 regulating rice tillering. In this review, we focus on the functions of miRNAs in controlling yield-related quantitative traits in rice, including panicle grain number, grain weight/size, panicle length and branching, tiller number per plant, spikelet number, seed setting rate, and leaf inclination, and discuss how to modulate the expression of these miRNAs using modern molecular biology tools to promote grain yield. This article is categorized under: RNA in Disease and Development > RNA in Development.

Perspectives on microRNAs and Phased Small Interfering RNAs in Maize (Zea mays L.): Functions and Big Impact on Agronomic Traits Enhancement.

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.

The interaction between miR160 and miR165/166 in the control of leaf development and drought tolerance in Arabidopsis.

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.

miR1432-OsACOT (Acyl-CoA thioesterase) module determines grain yield via enhancing grain filling rate in rice.

Rice grain filling rate contributes largely to grain productivity and accumulation of nutrients. MicroRNAs (miRNAs) are key regulators of development and physiology in plants and become a novel key target for engineering grain size and crop yield. However, there is little studies, so far, showing the miRNA regulation of grain filling and rice yield, in consequence. Here, we show that suppressed expression of rice miR1432 (STTM1432) significantly improves grain weight by enhancing grain filling rate and leads to an increase in overall grain yield up to 17.14% in a field trial. Molecular analysis identified rice Acyl-CoA thioesterase (OsACOT), which is conserved with ACOT13 in other species, as a major target of miR1432 by cleavage. Moreover, overexpression of miR1432-resistant form of OsACOT (OXmACOT) resembled the STTM1432 plants, that is, a large margin of an increase in grain weight up to 46.69% through improving the grain filling rate. Further study indicated that OsACOT was involved in biosynthesis of medium-chain fatty acids. In addition, RNA-seq based transcriptomic analyses of transgenic plants with altered expression of miR1432 demonstrated that downstream genes of miR1432-regulated network are involved in fatty acid metabolism and phytohormones biosynthesis and also overlap with the enrichment analysis of co-expressed genes of OsACOT, which is consistent with the increased levels of auxin and abscisic acid in STTM1432 and OXmACOT plants. Overall, miR1432-OsACOT module plays an important role in grain filling in rice, illustrating its capacity for engineering yield improvement in crops.

A Resource for Inactivation of MicroRNAs Using Short Tandem Target Mimic Technology in Model and Crop Plants.

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 Making of Leaves: How Small RNA Networks Modulate Leaf Development.

Leaf development is a sequential process that involves initiation, determination, transition, expansion and maturation. Many coding genes and a few non-coding small RNAs (sRNAs) have been identified as being involved in leaf development. sRNAs and their interactions not only determine gene expression and regulation, but also play critical roles in leaf development through their coordination with other genetic networks and physiological pathways. In this review, we first introduce the biogenesis pathways of sRNAs, mainly microRNAs (miRNAs) and trans-acting small interfering RNAs (ta-siRNAs), and then describe the function of miRNA-transcription factors in leaf development, focusing on guidance by interactive sRNA regulatory networks.

Suppression of microRNA159 impacts multiple agronomic traits in rice (Oryza sativa L.).

BACKGROUND: microRNAs (miRNAs) are important regulators in plant growth and development. miR159 is a conserved miRNA among different plant species and has various functions in plants. Studies on miR159 are mostly done on model plant, Arabidopsis thaliana. In rice, studies on miR159 were either based upon genome-wide expression analyses focused upon responses to different nitrogen forms and abiotic stress or upon phenotypic studies of transgenic plants overexpressing its precursor. STTM (Short Tandem Target Mimic) is an effective tool to block the activity of endogenous mature miRNA activity in plant. Therefore, specific roles of miR159 in rice could be explored by down regulating miR159 through STTM. RESULTS: In this study, expression of mature miR159 was successfully suppressed by STTM which resulted in the increased expressions of its two targets genes, OsGAMYB and OsGAMYBL1 (GAMYB-LIKE 1). Overall, STTM159 plants exhibited short stature along with smaller organ size and reduction in stem diameter, length of flag leaf, main panicle, spikelet hulls and grain size. Histological analysis of stem, leaf and mature spikelet hull showed the reduced number of small vascular bundles (SVB), less number of small veins (SV) between two big veins (LV) and less cell number in outer parenchyma. Gene Ontology (GO) enrichment analysis of differentially expressed genes between wild type plants and STTM159 transgenic plants showed that genes involved in cell division, auxin, cytokinin (CK) and brassinosteroids (BRs) biosynthesis and signaling are significantly down-regulated in STTM159 plants. CONCLUSION: Our data suggests that in rice, miR159 positively regulates organ size, including stem, leaf, and grain size due to the promotion of cell division. Further analysis from the RNA-seq data showed that the decreased cell divisions in STTM159 transgenic plants may result, at least partly from the lower expression of the genes involved in cell cycle and hormone homeostasis, which provides new insights of rice miR159-specific functions.

Knockdown of Rice microRNA166 by Short Tandem Target Mimic (STTM).

Small RNAs, including microRNAs (miRNAs), are abundant in plants and play key roles in controlling plant development and physiology. miRNAs regulate the expression of the target genes involved in key plant processes. Due to functional redundancy among miRNA family members in plants, an ideal approach to silence the expression of all members simultaneously, for their functional characterization, is desirable. Target mimic (TM) was the first approach to achieve this goal. Short tandem target mimic (STTM) is a potent approach complementing TM for silencing miRNAs in plants. STTMs have been successfully used in dicots to block miRNA functions. Here, we describe in detail the protocol for designing STTM construct to block miRNA functions in rice. Such approach can be applied to silence miRNAs in other monocots as well.

Silencing of Stress-Regulated miRNAs in Plants by Short Tandem Target Mimic (STTM) Approach.

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.

Essential RNA-Based Technologies and Their Applications in Plant Functional Genomics.

Genome sequencing has not only extended our understanding of the blueprints of many plant species but has also revealed the secrets of coding and non-coding genes. We present here a brief introduction to and personal account of key RNA-based technologies, as well as their development and applications for functional genomics of plant coding and non-coding genes, with a focus on short tandem target mimics (STTMs), artificial microRNAs (amiRNAs), and CRISPR/Cas9. In addition, their use in multiplex technologies for the functional dissection of gene networks is discussed.

To bloom or not to bloom: role of microRNAs in plant flowering.

During the course of their life cycles, plants undergo various morphological and physiological changes underlying juvenile-to-adult and adult-to-flowering phase transitions. To flower or not to flower is a key step of plasticity of a plant toward the start of its new life cycle. In addition to the previously revealed intrinsic genetic programs, exogenous cues, and endogenous cues, a class of small non-coding RNAs, microRNAs (miRNAs), plays a key role in plants making the decision to flower by integrating into the known flowering pathways. This review highlights the age-dependent flowering pathway with a focus on a number of timing miRNAs in determining such a key process. The contributions of other miRNAs which exist mainly outside the age pathway are also discussed. Approaches to study the flowering-determining miRNAs, their interactions, and applications are presented.

Functions of the poly(ADP-ribose) polymerase superfamily in plants.

Poly(ADP-ribosyl)ation is the covalent attachment of ADP-ribose subunits from NAD(+) to target proteins and was first described in plants in the 1970s. This post-translational modification is mediated by poly(ADP-ribose) polymerases (PARPs) and removed by poly(ADP-ribose) glycohydrolases (PARGs). PARPs have important functions in many biological processes including DNA repair, epigenetic regulation and transcription. However, these roles are not always associated with enzymatic activity. The PARP superfamily has been well studied in animals, but remains under-investigated in plants. Although plants lack the variety of PARP superfamily members found in mammals, they do encode three different types of PARP superfamily proteins, including a group of PARP-like proteins, the SRO family, that are plant specific. In plants, members of the PARP family and/or poly(ADP-ribosyl)ation have been linked to DNA repair, mitosis, innate immunity and stress responses. In addition, members of the SRO family have been shown to be necessary for normal sporophytic development. In this review, we summarize the current state of plant research into poly(ADP-ribosyl)ation and the PARP superfamily in plants.

RCD1 and SRO1 are necessary to maintain meristematic fate in Arabidopsis thaliana.

The radical-induced cell death1 and similar to RCD ONE1 genes of Arabidopsis thaliana encode members of the poly(ADP-ribose) polymerase (PARP) superfamily and have pleiotropic functions in development and abiotic stress response. In order to begin to understand the developmental and molecular bases of the defects seen in rcd1-3; sro1-1 plants, this study used the root as a model. Double mutant roots are short and display abnormally organized root apical meristems. However, acquisition of most cell fates within the root is not significantly disrupted. The identity of the quiescent centre is compromised, the zone of cell division is smaller than in wild-type roots and abnormal divisions are common, suggesting that RCD1 and SRO1 are necessary to maintain cells in a division-competent state and to regulate division plane placement. In addition, differentiation of several cell types is disrupted in rcd1-3; sro1-1 roots and shoots, demonstrating that RCD1 and SRO1 are also necessary for proper cell differentiation. Based on the data shown in this article and previous work, we hypothesize that RCD1 and SRO1 are involved in redox control and, in their absence, an altered redox balance leads to abnormal development.

Evolutionary history of the poly(ADP-ribose) polymerase gene family in eukaryotes.

BACKGROUND: The poly(ADP-ribose) polymerase (PARP) superfamily was originally identified as enzymes that catalyze the attachment of ADP-ribose subunits to target proteins using NAD+ as a substrate. The family is characterized by the catalytic site, termed the PARP signature. While these proteins can be found in a range of eukaryotes, they have been best studied in mammals. In these organisms, PARPs have key functions in DNA repair, genome integrity and epigenetic regulation. More recently it has been found that proteins within the PARP superfamily have altered catalytic sites, and have mono(ADP-ribose) transferase (mART) activity or are enzymatically inactive. These findings suggest that the PARP signature has a broader range of functions that initially predicted. In this study, we investigate the evolutionary history of PARP genes across the eukaryotes. RESULTS: We identified in silico 236 PARP proteins from 77 species across five of the six eukaryotic supergroups. We performed extensive phylogenetic analyses of the identified PARPs. They are found in all eukaryotic supergroups for which sequence is available, but some individual lineages within supergroups have independently lost these genes. The PARP superfamily can be subdivided into six clades. Two of these clades were likely found in the last common eukaryotic ancestor. In addition, we have identified PARPs in organisms in which they have not previously been described. CONCLUSIONS: Three main conclusions can be drawn from our study. First, the broad distribution and pattern of representation of PARP genes indicates that the ancestor of all extant eukaryotes encoded proteins of this type. Second, the ancestral PARP proteins had different functions and activities. One of these proteins was similar to human PARP1 and likely functioned in DNA damage response. The second of the ancestral PARPs had already evolved differences in its catalytic domain that suggest that these proteins may not have possessed poly(ADP-ribosyl)ation activity. Third, the diversity of the PARP superfamily is larger than previously documented, suggesting as more eukaryotic genomes become available, this gene family will grow in both number and type.