Conserved LBL1-ta-siRNA and miR165/166-RLD1/2 modules regulate root development in maize.

Root system architecture and anatomy of monocotyledonous maize is significantly different from dicotyledonous model Arabidopsis The molecular role of non-coding RNA (ncRNA) is poorly understood in maize root development. Here, we address the role of LEAFBLADELESS1 (LBL1), a component of maize trans-acting short-interfering RNA (ta-siRNA), in maize root development. We report that root growth, anatomical patterning, and the number of lateral roots (LRs), monocot-specific crown roots (CRs) and seminal roots (SRs) are significantly affected in lbl1-rgd1 mutant, which is defective in production of ta-siRNA, including tasiR-ARF that targets AUXIN RESPONSE FACTOR3 (ARF3) in maize. Altered accumulation and distribution of auxin, due to differential expression of auxin biosynthesis and transporter genes, created an imbalance in auxin signalling. Altered expression of microRNA165/166 (miR165/166) and its targets, ROLLED1 and ROLLED2 (RLD1/2), contributed to the changes in lbl1-rgd1 root growth and vascular patterning, as was evident by the altered root phenotype of Rld1-O semi-dominant mutant. Thus, LBL1/ta-siRNA module regulates root development, possibly by affecting auxin distribution and signalling, in crosstalk with miR165/166-RLD1/2 module. We further show that ZmLBL1 and its Arabidopsis homologue AtSGS3 proteins are functionally conserved.

Author Correction: Sirtinol, a Sir2 protein inhibitor, affects stem cell maintenance and root development in Arabidopsis thaliana by modulating auxin-cytokinin signaling components.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Auxin signaling modulates LATERAL ROOT PRIMORDIUM1 (LRP1) expression during lateral root development in Arabidopsis.

Auxin signaling mediated by various auxin/indole-3-acetic acid (Aux/IAAs) and AUXIN RESPONSE FACTORs (ARFs) regulate lateral root (LR) development by controlling the expression of downstream genes. LATERAL ROOT PRIMORDIUM1 (LRP1), a member of the SHORT INTERNODES/STYLISH (SHI/STY) family, was identified as an auxin-inducible gene. The precise developmental role and molecular regulation of LRP1 in root development remain to be understood. Here we show that LRP1 is expressed in all stages of LR development, besides the primary root. The expression of LRP1 is regulated by histone deacetylation in an auxin-dependent manner. Our genetic interaction studies showed that LRP1 acts downstream of auxin responsive Aux/IAAs-ARFs modules during LR development. We showed that auxin-mediated induction of LRP1 is lost in emerging LRs of slr-1 and arf7arf19 mutants roots. NPA treatment studies showed that LRP1 acts after LR founder cell specification and asymmetric division during LR development. Overexpression of LRP1 (LRP1 OE) showed an increased number of LR primordia (LRP) at stages I, IV and V, resulting in reduced emerged LR density, which suggests that it is involved in LRP development. Interestingly, LRP1-induced expression of YUC4, which is involved in auxin biosynthesis, contributes to the increased accumulation of endogenous auxin in LRP1 OE roots. LRP1 interacts with SHI, STY1, SRS3, SRS6 and SRS7 proteins of the SHI/STY family, indicating their possible redundant role during root development. Our results suggested that auxin and histone deacetylation affect LRP1 expression and it acts downstream of LR forming auxin response modules to negatively regulate LRP development by modulating auxin homeostasis in Arabidopsis thaliana.

Role of chromatin modification and remodeling in stem cell regulation and meristem maintenance in Arabidopsis.

In higher plants, pluripotent stem cells reside in the specialized microenvironment called stem cell niches (SCNs) harbored at the shoot apical meristem (SAM) and root apical meristem (RAM), which give rise to the aerial and underground parts of a plant, respectively. The model plant Arabidopsis thaliana (Arabidopsis) has been extensively studied to decipher the intricate regulatory mechanisms involving some key transcriptions factors and phytohormones that play pivotal roles in stem cell homeostasis, meristem maintenance, and organ formation. However, there is increasing evidence to show the epigenetic regulation of the chromatin architecture, gene expression exerting an influence on an innate balance between the self-renewal of stem cells, and differentiation of the progeny cells to a specific tissue type or organ. Post-translational histone modifications, ATP-dependent chromatin remodeling, and chromatin assembly/disassembly are some of the key features involved in the modulation of chromatin architecture. Here, we discuss the major epigenetic regulators and illustrate their roles in the regulation of stem cell activity, meristem maintenance, and related organ patterning in Arabidopsis.

Uncovering the molecular signature underlying the light intensity-dependent root development in Arabidopsis thaliana.

BACKGROUND: Root morphology is known to be affected by light quality, quantity and direction. Light signal is perceived at the shoot, translocated to roots through vasculature and further modulates the root development. Photoreceptors are differentially expressed in both shoot and root cells. The light irradiation to the root affects shoot morphology as well as whole plant development. The current work aims to understand the white light intensity dependent changes in root patterning and correlate that with the global gene expression profile. RESULTS: Different fluence of white light (WL) regulate overall root development via modulating the expression of a specific set of genes. Phytochrome A deficient Arabidopsis thaliana (phyA-211) showed shorter primary root compared to phytochrome B deficient (phyB-9) and wild type (WT) seedlings at a lower light intensity. However, at higher intensity, both mutants showed shorter primary root in comparison to WT. The lateral root number was observed to be lowest in phyA-211 at intensities of 38 and 75 µmol m - 2 s - 1. The number of adventitious roots was significantly lower in phyA-211 as compared to WT and phyB-9 under all light intensities tested. With the root phenotypic data, microarray was performed for four different intensities of WL light in WT. Here, we identified ~ 5243 differentially expressed genes (DEGs) under all light intensities. Gene ontology-based analysis indicated that different intensities of WL predominantly affect a subset of genes having catalytic activity and localized to the cytoplasm and membrane. Furthermore, when root is irradiated with different intensities of WL, several key genes involved in hormone, light signaling and clock-regulated pathways are differentially expressed. CONCLUSION: Using genome wide microarray-based approach, we have identified candidate genes in Arabidopsis root that responded to the changes in light intensities. Alteration in expression of genes such as PIF4, COL9, EPR1, CIP1, ARF18, ARR6, SAUR9, TOC1 etc. which are involved in light, hormone and clock pathway was validated by qRT-PCR. This indicates their potential role in light intensity mediated root development.

Whole mount in situ localization of miRNAs and target mRNA transcripts in plants.

The functional characterization of miRNAs often involves understanding of their spatiotemporal expression, which mostly relies on reporter-based or in situ hybridization studies. The available in situ localization methods follow separate protocols for pre-hybridization, hybridization, post-hybridization, and detection steps for both miRNA and mRNA transcripts in plants. In this study, we present a single method which can be used for whole mount in situ localization of both miRNAs and mRNAs in different plant tissues. Our modified method provides enhanced sensitivity for the localization of miRNA and their target transcripts. Consequently, a less laborious, time-saving, economic and efficient method has been proposed by the modification of pre-hybridization, hybridization, post-hybridization and detection steps.

Improved Method of RNA Isolation from Laser Capture Microdissection (LCM)-Derived Plant Tissues.

Laser capture microdissection (LCM) is a tool to isolate desired and/or less accessible cells or tissues from a heterogeneous population. In the current method, we describe an efficient and cost-effective method to obtain both high-quality mRNA and miRNAs in sufficient quantity from LCM-derived plant tissues. The quality of the isolated RNA can be assessed using Bioanalyzer. Using modified stem-loop RT-PCR, we confirmed the presence of 21-24 nucleotide (nt) long mature miRNAs. This modified LCM-based method has been found to be suitable for the tissue-specific expression analysis of both genes and small RNAs (miRNAs).

Plant small RNAs: advancement in the understanding of biogenesis and role in plant development.

MAIN CONCLUSION: Present review addresses the advances made in the understanding of biogenesis of plant small RNAs and their role in plant development. We discuss the elaborate role of microRNAs (miRNAs) and trans-acting small interfering RNAs (ta-siRNAs) in various aspects of plant growth and development and highlight relevance of small RNA mobility. Small non-coding RNAs regulate various aspects of plant development. Small RNAs (sRNAs) of 21-24 nucleotide length are derived from double-stranded RNAs through the combined activity of several biogenesis and processing components. These sRNAs function by negatively regulating the expression of target genes. miRNAs and ta-siRNAs constitute two important classes of endogenous small RNAs in plants, which play important roles in plant growth and developmental processes like embryogenesis, organ formation and patterning, shoot and root growth, and reproductive development. Biogenesis of miRNAs is a multistep process which includes transcription, processing and modification, and their loading onto RNA-induced silencing complex (RISC). RISC-loaded miRNAs carry out post-transcriptional silencing of their target(s). Recent studies identified orthologues of different biogenesis components of novel and conserved small RNAs from different model plants. Although many small RNAs have been identified from diverse plant species, only a handful of them have been functionally characterized. In this review, we discuss the advances made in understanding the biogenesis, functional conservation/divergence in miRNA-mediated gene regulation, and the developmental role of small RNAs in different plant species.

Phytohormonal crosstalk modulates the expression of miR166/165s, target Class III HD-ZIPs, and KANADI genes during root growth in Arabidopsis thaliana.

Both phytohormones and non-coding microRNAs (miRNAs) play important role in root development in Arabidopsis thaliana. Mature miR166/165 s, which are derived from precursor transcripts of concerned genes, regulate developmental processes, including leaf and root patterning, by targeting Class III HOMEODOMAIN LEUCINE-ZIPPER (HD-ZIP III) transcription factors (TFs). However, their regulation through hormones remained poorly understood. Here, we show that several phytohormones dynamically regulate the spatio-temporal expression pattern of miR166/165 and target HD-ZIP IIIs in developing roots. Hormone signaling pathway mutants show differential expression pattern of miR166/165, providing further genetic evidence for multilayered regulation of these genes through phytohormones. We further show that a crosstalk of at least six different phytohormones regulate the miR166/165, their target HD-ZIP IIIs, and KANADI (KANs). Our results suggest that HD-ZIP IIIs mediated root development is modulated both transcriptionally through phytohormones and KANs, and post-transcriptionally by miR166/165 that in turn are also regulated by the phytohormonal crosstalk.

Sirtinol, a Sir2 protein inhibitor, affects stem cell maintenance and root development in Arabidopsis thaliana by modulating auxin-cytokinin signaling components.

In Arabidopsis thaliana, besides several key transcription factors and chromatin modifiers, phytohormones auxin and cytokinin play pivotal role in shoot and root meristem maintenance, and lateral root (LR) development. Sirtinol, a chemical inhibitor of Sir2 proteins, is known to promote some auxin induced phenotypes in Arabidopsis. However, its effect on plant stem cell maintenance or organ formation remained unaddressed. Here we show that sirtinol affects meristem maintenance by altering the expression of key stem cell regulators, cell division and differentiation by modulating both auxin and cytokinin signaling in Arabidopsis thaliana. The expression of shoot stem cell niche related genes WUSCHEL (WUS) and CLAVATA3 (CLV3) was upregulated, whereas SHOOT MERISTEMLESS (STM) was downregulated in sirtinol treated seedlings. The expression level and domain of key root stem cell regulators PLETHORA (PLTs) and WUS-Related Homeobox 5 (WOX5) were altered in sirtinol treated roots. Sirtinol affects LR development by disturbing proper auxin transport and maxima formation, similar to 2,4-dichlorophenoxyacetic acid (2,4-D). Sirtinol also affects LR formation by altering cytokinin biosynthesis and signaling genes in roots. Therefore, sirtinol affects shoot and root growth, meristem maintenance and LR development by altering the expression of cytokinin-auxin signaling components, and regulators of stem cells, meristems, and LRs.

An Efficient LCM-Based Method for Tissue Specific Expression Analysis of Genes and miRNAs.

Laser Capture Microdissection (LCM) is a powerful tool to isolate and study gene expression pattern of desired and less accessible cells or tissues from a heterogeneous population. Existing LCM-based methods fail to obtain high quality RNA including small RNAs from small microdissected plant tissue and therefore, are not suitable for miRNA expression studies. Here, we describe an efficient and cost-effective method to obtain both high quality RNA and miRNAs from LCM-derived embryonic root apical meristematic tissue, which is difficult to access. We have significantly modified and improved the tissue fixation, processing, sectioning and RNA isolation steps and minimized the use of kits. Isolated RNA was checked for quality with bioanalyzer and used for gene expression studies. We have confirmed the presence of 19-24 nucleotide long mature miRNAs using modified stem-loop RT-PCR. This modified LCM-based method is suitable for tissue specific expression analysis of both genes and small RNAs (miRNAs).

Coevolution Pattern and Functional Conservation or Divergence of miR167s and their targets across Diverse Plant Species.

microRNAs (miRNAs), a class of endogenously produced small non-coding RNAs of 20-21 nt length, processed from precursor miRNAs, regulate many developmental processes by negatively regulating the target genes in both animals and plants. The coevolutionary pattern of a miRNA family and their targets underscores its functional conservation or diversification. The miR167 regulates various aspects of plant development in Arabidopsis by targeting ARF6 and ARF8. The evolutionary conservation or divergence of miR167s and their target genes are poorly understood till now. Here we show the evolutionary relationship among 153 MIR167 genes obtained from 33 diverse plant species. We found that out of the 153 of miR167 sequences retrieved from the "miRBase", 27 have been annotated to be processed from the 3' end, and have diverged distinctively from the other miR167s produced from 5' end. Our analysis reveals that gma-miR167h/i and mdm-miR167a are processed from 3' end and have evolved separately, diverged most resulting in novel targets other than their known ones, and thus led to functional diversification, especially in apple and soybean. We also show that mostly conserved miR167 sequences and their target AUXIN RESPONSE FACTORS (ARFs) have gone through parallel evolution leading to functional diversification among diverse plant species.

Balanced activity of microRNA166/165 and its target transcripts from the class III homeodomain-leucine zipper family regulates root growth in Arabidopsis thaliana.

Overexpression of miR166/165 down-regulates target HD - ZIP IIIs and promotes root growth by enhancing cell division and meristematic activity, whereas overexpression of HD - ZIP IIIs inhibits root growth in Arabidopsis thaliana. Post-embryonic growth of higher plants is maintained by active meristems harbouring undifferentiated cells. Shoot and root apical meristems (SAM and RAM) utilize both similar and distinct signalling mechanisms for their maintenance in Arabidopsis thaliana. An important regulatory role in this context has the interaction of microRNAs with their target mRNAs, mostly encoding transcription factors. One class of microRNA166/165 (miR166/165) has been implicated in the maintenance of SAM and vascular patterning. Here, we show that miR166/165 plays an important role in root growth also by negatively regulating its target transcripts, HD-ZIP IIIs, in the RAM. While overexpression of miR166 promotes RAM activity, overexpression of its targets reduces RAM activity. These results reveal a conserved role of miR166/165 in the maintenance of SAM and RAM activity in A. thaliana.

SWP1 negatively regulates lateral root initiation and elongation in Arabidopsis.

The main root and continuously emerging lateral roots constitute the root architecture of an adult plant during its postembryonic development. Epigenetic modifications like methylation or deacetylation of histones have been suggested to regulate root development. SWP1/LDL1, a component of plant specific corepressor complex, has been implicated in the induction of flowers and root through histone modifications in Arabidopsis. However, molecular role of SWP1 in regulating the lateral root development remained unexplored. Here we show that SWP1 regulates lateral root initiation and elongation in Arabidopsis. Mutation in SWP1 increases both the density and length of lateral roots. SWP1 negatively regulates lateral root initiation through direct/indirect transcriptional repression of lateral root promoting factors, such as AUXIN RESPONSE FACTORS (ARFs) and GATA23.

Percutaneous coronary intervention for myocardial infarction during pregnancy: a new trend?

Recent studies report that American women are increasingly delaying their first births. While the proportion of births in older women has been increasing, there is also increased prevalence of cardiovascular risk factors and complications of pregnancy with increasing maternal age. We present 2 cases of acute myocardial infarction occurring during pregnancy. The mothers were both over 35 years old, and had significant risk factors for coronary disease. Both were found to have atherosclerotic coronary lesions, and were managed with coronary intervention with successful reperfusion. One woman successfully delivered a healthy infant at term. The other had a spontaneous abortion shortly after discharge from the hospital. Given current demographic trends, it is likely that such cases will be more commonly seen.