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1.  Enhancement of Indole-3-Acetic Acid Photodegradation by Vitamin B6 
Molecular Plant  2013;6(6):1992-1995.
PMCID: PMC3834970  PMID: 23723155
3.  Age-Triggered and Dark-Induced Leaf Senescence Require the bHLH Transcription Factors PIF3, 4, and 5 
Molecular Plant  2014;7(12):1776-1787.
Numerous lines of evidence have suggested an involvement of phytochromes in the regulation of leaf senescence, but the related signaling pathway and physiological mechanisms are poorly understood. In this study, we demonstrated that PIF3, 4, and 5, the master regulators of light signaling, modulate age-triggered and dark-induced senescence and particularly PIF4 positively regulates leaf senescence by directly targeting genes related to chlorophyll degradation and chloroplast activity maintaining as well as ethylene biosynthesis.
Leaf senescence can be triggered and promoted by a large number of developmental and environmental factors. Numerous lines of evidence have suggested an involvement of phytochromes in the regulation of leaf senescence, but the related signaling pathways and physiological mechanisms are poorly understood. In this study, we initially identified phytochrome-interacting factors (PIFs) 3, 4, and 5 as putative mediators of leaf senescence. Mutations of the PIF genes resulted in a significantly enhanced leaf longevity in age-triggered and dark-induced senescence, whereas overexpressions of these genes accelerated age-triggered and dark-induced senescence in Arabidopsis. Consistently, loss-of-function of PIF4 attenuated dark-induced transcriptional changes associated with chloroplast deterioration and reactive oxygen species (ROS) generation. ChIP–PCR and Dual-Luciferase assays demonstrated that PIF4 can activate chlorophyll degradation regulatory gene NYE1 and repress chloroplast activity maintainer gene GLK2 by binding to their promoter regions. Finally, dark-induced ethylene biosynthesis and ethylene-induced senescence were both dampened in pif4, suggesting the involvement of PIF4 in both ethylene biosynthesis and signaling pathway. Our study provides evidence that PIF3, 4, and 5 are novel positive senescence mediators and gains an insight into the mechanism of light signaling involved in the regulation of leaf senescence.
PMCID: PMC4261840  PMID: 25296857
phytochrome-interacting factor (PIF); leaf senescence; chloroplast deterioration; NYE1/SGR1; GLK2; ethylene.
4.  Splicing of Receptor-Like Kinase-Encoding SNC4 and CERK1 is Regulated by Two Conserved Splicing Factors that Are Required for Plant Immunity 
Molecular Plant  2014;7(12):1766-1775.
Two conserved splicing factors, SUA and RSN2, were identified from a suppressor screen of snc4-1D. Both are required for alternative splicing of receptor-like kinase (RLK)-encoding SNC4 and CERK1 and their functions, suggesting that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1.
Plant immune receptors belonging to the receptor-like kinase (RLK) family play important roles in the recognition of microbial pathogens and activation of downstream defense responses. The Arabidopsis mutant snc4-1D contains a gain-of-function mutation in the RLK SNC4 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE4), which leads to constitutive activation of defense responses. Analysis of suppressor mutants of snc4-1D identified two conserved splicing factors, SUA (SUPPRESSOR OF ABI3-5) and RSN2 (REQUIRED FOR SNC4-1D 2), that are required for the constitutive defense responses in snc4-1D. In sua and rsn2 mutants, SNC4 splicing is altered and the amount of SNC4 transcripts is reduced. Further analysis showed that SUA and RSN2 are also required for the proper splicing of CERK1 (CHITIN ELICITOR RECEPTOR KINASE1), which encodes another RLK that functions as a receptor for chitin. In sua and rsn2 mutants, induction of reactive oxygen species by chitin is reduced and the non-pathogenic bacteria Pseudomonas syringae pv. tomato DC3000hrcC grows to higher titers than in wild-type plants. Our study suggests that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1.
PMCID: PMC4261838  PMID: 25267732
plant immunity; receptor-like kinase; alternative splicing; SUA; RSN2; SNC4; CERK1.
5.  SPINDLY, ERECTA, and Its Ligand STOMAGEN Have a Role in Redox-Mediated Cortex Proliferation in the Arabidopsis Root 
Molecular Plant  2014;7(12):1727-1739.
Reactive oxygen species (ROS) are signaling molecules, but how they are perceived in plants remains unclear. This study showed that cortex proliferation in the Arabidopsis root can be induced by hydrogen peroxide and that the receptor kinase ERECTA and one of its ligands, STOMAGEN, are involved in a signaling pathway that couples ROS sensing with redox-mediated cortex proliferation. This study also revealed a new role for SPINDLY (SPY), a putative O-GlcNAc transferase, in cellular redox homeostasis.
Reactive oxygen species (ROS) are harmful to all living organisms and therefore they must be removed to ensure normal growth and development. ROS are also signaling molecules, but so far little is known about the mechanisms of ROS perception and developmental response in plants. We here report that hydrogen peroxide induces cortex proliferation in the Arabidopsis root and that SPINDLY (SPY), an O-linked glucosamine acetyltransferase, regulates cortex proliferation by maintaining cellular redox homeostasis. We also found that mutation in the leucine-rich receptor kinase ERECTA and its putative peptide ligand STOMAGEN block the effect of hydrogen peroxide on root cortex proliferation. However, ERECTA and STOMAGEN are expressed in the vascular tissue, whereas extra cortex cells are produced from the endodermis, suggesting the involvement of intercellular signaling. SPY appears to act downstream of ERECTA, because the spy mutation still caused cortex proliferation in the erecta mutant background. We therefore have not only gained insight into the mechanism by which SPY regulates root development but also uncovered a novel pathway for ROS signaling in plants. The importance of redox-mediated cortex proliferation as a protective mechanism against oxidative stress is also discussed.
PMCID: PMC4261839  PMID: 25267734
SPY; ERECTA; STOMAGEN; redox homeostasis; ROS signaling; abiotic stress; cortex proliferation; Arabidopsis thaliana.
6.  Genome-Wide Analysis of Histone Modifications: H3K4me2, H3K4me3, H3K9ac, and H3K27ac in Oryza sativa L. Japonica 
Molecular Plant  2013;6(5):1463-1472.
H3K4me2/3, H3K9ac, and H3K27ac investigated by ChIP-Seq showed enrichment in generic regions and transcription start sites, and associated with active transcription in rice. They were used to discover unannotated genes and to predict transcription factor binding sites together with DNase-Seq data.
While previous studies have shown that histone modifications could influence plant growth and development by regulating gene transcription, knowledge about the relationships between these modifications and gene expression is still limited. This study used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq), to investigate the genome-wide distribution of four histone modifications: di and trimethylation of H3K4 (H3K4me2 and H3K4me3) and acylation of H3K9 and H3K27 (H3K9ac and H3K27ac) in Oryza sativa L. japonica. By analyzing published DNase-Seq data, this study explored DNase-Hypersensitive (DH) sites along the rice genome. The histone marks appeared mainly in generic regions and were enriched around the transcription start sites (TSSs) of genes. This analysis demonstrated that the four histone modifications and the DH sites were all associated with active transcription. Furthermore, the four histone modifications were highly concurrent with transcript regions—a promising feature that was used to predict missing genes in the rice gene annotation. The predictions were further validated by experimentally confirming the transcription of two predicted missing genes. Moreover, a sequence motif analysis was constructed in order to identify the DH sites and many putative transcription factor binding sites.
PMCID: PMC3842134  PMID: 23355544
bioinformatics; chromatin structure and remodeling; epigenetics; gene regulation; genomics; rice.
7.  1O2-Mediated and EXECUTER-Dependent Retrograde Plastid-to-Nucleus Signaling in Norflurazon-Treated Seedlings of Arabidopsis thaliana  
Molecular Plant  2013;6(5):1580-1591.
Seedlings of Arabidopsis have been exposed to norflurazon. Depending on the developmental stage at which seedlings were first exposed to the inhibitor, enhanced production of reactive oxygen species occurred and, among others, 1O2-mediated and EXECUTER-dependent retrograde signaling was induced.
Chloroplast development depends on the synthesis and import of a large number of nuclear-encoded proteins. The synthesis of some of these proteins is affected by the functional state of the plastid via a process known as retrograde signaling. Retrograde plastid-to-nucleus signaling has been often characterized in seedlings of Arabidopsis thaliana exposed to norflurazon (NF), an inhibitor of carotenoid biosynthesis. Results of this work suggested that, throughout seedling development, a factor is released from the plastid to the cytoplasm that indicates a perturbation of plastid homeostasis and represses nuclear genes required for normal chloroplast development. The identity of this factor is still under debate. Reactive oxygen species (ROS) were among the candidates discussed as possible retrograde signals in NF-treated plants. In the present work, this proposed role of ROS has been analyzed. In seedlings grown from the very beginning in the presence of NF, ROS-dependent signaling was not detectable, whereas, in seedlings first exposed to NF after light-dependent chloroplast formation had been completed, enhanced ROS production occurred and, among others, 1O2-mediated and EXECUTER-dependent retrograde signaling was induced. Hence, depending on the developmental stage at which plants are exposed to NF, different retrograde signaling pathways may be activated, some of which are also active in non-treated plants under light stress.
PMCID: PMC3842135  PMID: 23376773
retrograde signaling; singlet oxygen; Executer; norflurazon; programmed cell death; photo-oxidative stress.
9.  MADS-Box Transcription Factor AGL21 Regulates Lateral Root Development and Responds to Multiple External and Physiological Signals 
Molecular Plant  2014;7(11):1653-1669.
MADS-box transcription factor AGL21 is responsive to several phytohormones as well as environmental cues and positively regulates auxin accumulation in lateral root primordia and lateral roots by enhancing local auxin biosynthesis, thus stimulating lateral root initiation and growth. Therefore, AGL21 may be involved in various environmental and physiological signals-mediated lateral root development.
Plant root system morphology is dramatically influenced by various environmental cues. The adaptation of root system architecture to environmental constraints, which mostly depends on the formation and growth of lateral roots, is an important agronomic trait. Lateral root development is regulated by the external signals coordinating closely with intrinsic signaling pathways. MADS-box transcription factors are known key regulators of the transition to flowering and flower development. However, their functions in root development are still poorly understood. Here we report that AGL21, an AGL17-clade MADS-box gene, plays a crucial role in lateral root development. AGL21 was highly expressed in root, particularly in the root central cylinder and lateral root primordia. AGL21 overexpression plants produced more and longer lateral roots while agl21 mutants showed impaired lateral root development, especially under nitrogen-deficient conditions. AGL21 was induced by many plant hormones and environmental stresses, suggesting a function of this gene in root system plasticity in response to various signals. Furthermore, AGL21 was found positively regulating auxin accumulation in lateral root primordia and lateral roots by enhancing local auxin biosynthesis, thus stimulating lateral root initiation and growth. We propose that AGL21 may be involved in various environmental and physiological signals-mediated lateral root development and growth.
PMCID: PMC4228986  PMID: 25122697
MADS; root system architecture; lateral root; AGL21; auxin; nitrate; sulfate.
10.  Timing Is Everything: Highly Specific and Transient Expression of a MAP Kinase Determines Auxin-Induced Leaf Venation Patterns in Arabidopsis  
Molecular Plant  2014;7(11):1637-1652.
The Arabidopsis MAP kinase AtMPK10 has long been considered as a pseudo-gene without visible function for the plant. Here we show that AtMPK10 is functional only in a very narrow time window in leaves at sites of local auxin maxima where it regulates leaf venation complexity together with the upstream kinase AtMKK2.
Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules present in all eukaryotes. In plants, MAPK cascades were shown to regulate cell division, developmental processes, stress responses, and hormone pathways. The subgroup A of Arabidopsis MAPKs consists of AtMPK3, AtMPK6, and AtMPK10. AtMPK3 and AtMPK6 are activated by their upstream MAP kinase kinases (MKKs) AtMKK4 and AtMKK5 in response to biotic and abiotic stress. In addition, they were identified as key regulators of stomatal development and patterning. AtMPK10 has long been considered as a pseudo-gene, derived from a gene duplication of AtMPK6. Here we show that AtMPK10 is expressed highly but very transiently in seedlings and at sites of local auxin maxima leaves. MPK10 encodes a functional kinase and interacts with the upstream MAP kinase kinase (MAPKK) AtMKK2. mpk10 mutants are delayed in flowering in long-day conditions and in continuous light. Moreover, cotyledons of mpk10 and mkk2 mutants have reduced vein complexity, which can be reversed by inhibiting polar auxin transport (PAT). Auxin does not affect AtMPK10 expression while treatment with the PAT inhibitor HFCA extends the expression in leaves and reverses the mpk10 mutant phenotype. These results suggest that the AtMKK2–AtMPK10 MAPK module regulates venation complexity by altering PAT efficiency.
PMCID: PMC4228985  PMID: 25064848
Arabidopsis MAP kinase; leaf development; polar auxin transport; leaf venation pattern.
11.  Characterization and DNA-Binding Specificities of Ralstonia TAL-Like Effectors 
Molecular Plant  2013;6(4):1318-1330.
We report the characterization of three Ralstonia TAL-like effectors, which mediate DNA binding and can be used as customizable architectures for DNA targeting. We determined DNA-binding specificities of novel repeat variable di-residues (RVDs) and devised a repeat assembly approach for engineering Ralstonia solanacearum TALE-like proteins (RTLs).
Transcription activator-like effectors (TALEs) from Xanthomonas sp. have been used as customizable DNA-binding modules for genome-engineering applications. Ralstonia solanacearum TALE-like proteins (RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts specifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.
PMCID: PMC3716395  PMID: 23300258
Ralstonia solanacearum; genome engineering; TAL effectors; TALE activators and repressors; TALE nucleases (TALENs); targeted genome modifications
12.  Signaling in Pollen Tube Growth: Crosstalk, Feedback, and Missing Links 
Molecular Plant  2013;6(4):1053-1064.
Pollen tubes represent an attractive model system to investigate polarized cell growth. We will discuss the current state of play of knowledge of the regulatory roles of signaling networks in the cellular activities of the pollen tube tip.
Pollen tubes elongate rapidly at their tips through highly polarized cell growth known as tip growth. Tip growth requires intensive exocytosis at the tip, which is supported by a dynamic cytoskeleton and vesicle trafficking. Several signaling pathways have been demonstrated to coordinate pollen tube growth by regulating cellular activities such as actin dynamics, exocytosis, and endocytosis. These signaling pathways crosstalk to form a signaling network that coordinates the cellular processes required for tip growth. The homeostasis of key signaling molecules is critical for the proper elongation of the pollen tube tip, and is commonly fine-tuned by positive and negative regulations. In addition to the major signaling pathways, emerging evidence implies the roles of other signals in the regulation of pollen tube growth. Here we review and discuss how these signaling networks modulate the rapid growth of pollen tubes.
PMCID: PMC3842152  PMID: 23873928
polarity; pollen development; reproductive biology; signal transduction.
13.  AtPRK2 Promotes ROP1 Activation via RopGEFs in the Control of Polarized Pollen Tube Growth 
Molecular Plant  2012;6(4):1187-1201.
The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho guanine nucleotide exchange factors (RopGEFs) and in turn may be activated by an unknown factor through releasing RopGEF1’s auto-inhibition. In this study, we found that RopGEF1 forms a complex with ROP1 and AtPRK2, a receptor-like protein kinase previously shown to interact with RopGEFs. AtPRK2 phosphorylated RopGEF1 in vitro and the atprk1,2,5 triple mutant showed defective pollen tube growth, similar to the phenotype of the ropgef1,9,12,14 quadruple mutant. Overexpression of a dominant negative form of AtPRK2 (DN-PRK2) inhibited pollen germination in Arabidopsis and reduced pollen elongation in tobacco. The DN-PRK2-induced pollen germination defect was rescued by overexpressing a constitutively active form of RopGEF1, RopGEF1(90–457), implying that RopGEF1 acts downstream of AtPRK2. Moreover, AtPRK2 increased ROP1 activity at the apical plasma membrane whereas DN-PRK2 reduced ROP1 activity. Finally, two mutations at the C-terminal putative phosphorylation sites of RopGEF1 (RopGEF1S460A and RopGEF1S480A) eliminated the function of RopGEF1 in vivo. Taken together, our results support the hypothesis that AtPRK2 acts as a positive regulator of the ROP1 signaling pathway most likely by activating RopGEF1 through phosphorylation.
PMCID: PMC3888354  PMID: 23024212
AtPRK2; RopGEF1; ROP GTPase; auto-inhibition; polarity growth.
15.  Control of Cell Wall Extensibility during Pollen Tube Growth 
Molecular Plant  2013;6(4):998-1017.
Tip-growing pollen tubes achieve rapid elongation while maintaining cell wall integrity by balancing local expansion, controlled by local changes in wall viscosity, against exocytosis, influenced by the activity of the actin cytoskeleton, cellular energetics, and calcium and proton physiology.
In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expansion rate is controlled by local changes in the viscosity of the apical wall. We focus on several different structures and underlying processes that are thought to be major participants including exocytosis, the organization and activity of the actin cytoskeleton, calcium and proton physiology, and cellular energetics. We think that the actin cytoskeleton, in particular the apical cortical actin fringe, directs the flow of vesicles to the apical domain, where they fuse with the plasma membrane and contribute their contents to the expanding cell wall. While pH gradients, as generated by a proton-ATPase located on the plasma membrane along the side of the clear zone, may regulate rapid actin turnover and new polymerization in the fringe, the tip-focused calcium gradient biases secretion towards the polar axis. The recent data showing that exocytosis of new wall material precedes and predicts the process of cell elongation provide support for the idea that the intussusception of newly secreted pectin contributes to decreases in apical wall viscosity and to cell expansion. Other prime factors will be the localization and activity of the enzyme pectin methyl-esterase, and the chelation of calcium by pectic acids. Finally, we acknowledge a role for reactive oxygen species in the control of wall viscosity.
PMCID: PMC4043104  PMID: 23770837
cell expansion; cell walls; cytoskeleton dynamics; polarity; pollen development.
16.  OPT3 Is a Component of the Iron-Signaling Network between Leaves and Roots and Misregulation of OPT3 Leads to an Over-Accumulation of Cadmium in Seeds 
Molecular Plant  2014;7(9):1455-1469.
Long-distance communication between leaves and roots are key to properly regulate the uptake of trace metals from the soil. The molecular basis of this shoot-to-root signaling is currently unknown. In this manuscript, we describe the role of OPT3 in the shoot-to-root signaling of the iron status in Arabidopsis. We also show that reduced expression of OPT3 induces an over-accumulation of the toxic metal cadmium, but not other metals, in seeds.
Plants and seeds are the main dietary sources of zinc, iron, manganese, and copper, but are also the main entry point for toxic elements such as cadmium into the food chain. We report here that an Arabidopsis oligopeptide transporter mutant, opt3-2, over-accumulates cadmium (Cd) in seeds and roots but, unexpectedly, under-accumulates Cd in leaves. The cadmium distribution in opt3-2 differs from iron, zinc, and manganese, suggesting a metal-specific mechanism for metal partitioning within the plant. The opt3-2 mutant constitutively up-regulates the Fe/Zn/Cd transporter IRT1 and FRO2 in roots, indicative of an iron-deficiency response. No genetic mutants that impair the shoot-to-root signaling of iron status in leaves have been identified. Interestingly, shoot-specific expression of OPT3 rescues the Cd sensitivity and complements the aberrant expression of IRT1 in opt3-2 roots, suggesting that OPT3 is required to relay the iron status from leaves to roots. OPT3 expression was found in the vasculature with preferential expression in the phloem at the plasma membrane. Using radioisotope experiments, we found that mobilization of Fe from leaves is severely affected in opt3-2, suggesting that Fe mobilization out of leaves is required for proper trace-metal homeostasis. When expressed in yeast, OPT3 does not localize to the plasma membrane, precluding the identification of the OPT3 substrate. Our in planta results show that OPT3 is important for leaf phloem-loading of iron and plays a key role regulating Fe, Zn, and Cd distribution within the plant. Furthermore, ferric chelate reductase activity analyses provide evidence that iron is not the sole signal transferred from leaves to roots in leaf iron status signaling.
PMCID: PMC4153440  PMID: 24880337
phloem transport; seed loading; metal homeostasis; iron deficiency; ionomics.
17.  COP1 Jointly Modulates Cytoskeletal Processes and Electrophysiological Responses Required for Stomatal Closure 
Molecular Plant  2014;7(9):1441-1454.
COP1-mediated proteolysis is required for stomatal closure. In guard cells, COP1 function is linked to microtubule destabilization and the activity of S-type anion channels leading to stomatal closure.
Reorganization of the cortical microtubule cytoskeleton is critical for guard cell function. Here, we investigate how environmental and hormonal signals cause these rearrangements and find that COP1, a RING-finger-type ubiquitin E3 ligase, is required for degradation of tubulin, likely by the 26S proteasome. This degradation is required for stomatal closing. In addition to regulating the cytoskeleton, we show that cop1 mutation impaired the activity of S-type anion channels, which are critical for stomatal closure. Thus, COP1 is revealed as a potential coordinator of cytoskeletal and electrophysiological activities required for guard cell function.
PMCID: PMC4153439  PMID: 25151660
stomatal function; microtubule dynamics; hormone signaling.
18.  LSCHL4 from Japonica Cultivar, Which Is Allelic to NAL1, Increases Yield of Indica Super Rice 93-11 
Molecular Plant  2014;7(8):1350-1364.
The basic premise of high yield in rice is to improve leaf photosynthetic efficiency, and coordinate the source–sink relationship in rice plants. The quantitative trait loci (QTLs) qLSCHL4, japonica NAL1 allele from Nipponbare has a pleiotropic function, effectively increased leaf chlorophyll content, enlarged flag leaf size, and enhanced the yield of indica rice cultivar.
The basic premise of high yield in rice is to improve leaf photosynthetic efficiency and coordinate the source–sink relationship in rice plants. Quantitative trait loci (QTLs) related to morphological traits and chlorophyll content of rice leaves were detected at the stages of heading to maturity, and a major QTL (qLSCHL4) related to flag leaf shape and chlorophyll content was detected at both stages in recombinant inbred lines constructed using the indica rice cultivar 93-11 and the japonica rice cultivar Nipponbare. Map-based cloning and expression analysis showed that LSCHL4 is allelic to NAL1, a gene previously reported in narrow leaf mutant of rice. Overexpression lines transformed with vector carrying LSCHL4 from Nipponbare and a near-isogenic line of 93-11 (NIL-9311) had significantly increased leaf chlorophyll content, enlarged flag leaf size, and improved panicle type. The average yield of NIL-9311 was 18.70% higher than that of 93-11. These results indicate that LSCHL4 had a pleiotropic function. Exploring and pyramiding more high-yield alleles resembling LSCHL4 for super rice breeding provides an effective way to achieve new breakthroughs in raising rice yield and generate new ideas for solving the problem of global food safety.
PMCID: PMC4115278  PMID: 24795339
rice breeding; QTL; qLSCHL4; panicle type; pleiotropism; yield potential.
21.  Mechanisms of Small RNA Generation from Cis-NATs in Response to Environmental and Developmental Cues 
Molecular Plant  2013;6(3):704-715.
This review summarizes the current findings on the occurrence and characteristics of cis-natural antisense transcripts (NATs) and nat-siRNAs; discusses the biogenesis, regulations and functions of nat-siRNAs; and highlights the advantages and limitations of new technologies on cis-NATs detection.
A large proportion of eukaryotic genomes is transcribed from both positive and negative strands of DNA and thus may generate overlapping sense and antisense transcripts. Some of these so-called natural antisense transcripts (NATs) are possibly co-regulated. When the overlapping sense and antisense transcripts are expressed at the same time in the same cell in response to various developmental and environmental cues; they may form double-stranded RNAs, which could be recognized by the small RNA biogenesis machinery and processed into small interfering RNAs (siRNAs). cis-NAT-derived siRNAs (nat-siRNAs) are present in plants, animals, and fungi. In plants, the presence of nat-siRNAs is supported not only by Northern blot and genetic analyses, but also by the fact that there is an overall sixfold enrichment of siRNAs in the overlapping regions of cis-NATs and 19%–29% of the siRNA-generating cis-NATs in plants give rise to siRNAs only in their overlapping regions. Silencing mediated by nat-siRNAs is one of the mechanisms for regulating the expression of the cis-NATs. This review focuses on challenging issues related to the biogenesis mechanisms as well as regulation and detection of nat-siRNAs. The advantages and limitations of new technologies for detecting cis-NATs, including direct RNA sequencing and strand-specific RNA sequencing, are also discussed.
PMCID: PMC3660955  PMID: 23505223
gene expression; gene regulation; gene silencing.
22.  A Global Identification and Analysis of Small Nucleolar RNAs and Possible Intermediate-Sized Non-Coding RNAs in Oryza sativa  
Molecular Plant  2012;6(3):830-846.
Accumulating evidence suggests that non-coding RNAs (ncRNAs) are both widespread and functionally important in many eukaryotic organisms. In this study, we employed a special size fractionation and cDNA library construction method followed by 454 deep sequencing to systematically profile rice intermediate-size ncRNAs. Our analysis resulted in the identification of 1349 ncRNAs in total, including 754 novel ncRNAs of an unknown functional category. Chromosome distribution of all identified ncRNAs showed no strand bias, and displayed a pattern similar to that observed in protein-coding genes with few chromosome dependencies. More than half of the ncRNAs were centered around the plus-strand of the 5’ and 3’ termini of the coding regions. The majority of the novel ncRNAs were rice specific, while 78% of the small nucleolar RNAs (snoRNAs) were conserved. Tandem duplication drove the expansion of over half of the snoRNA gene families. Furthermore, 90% of the snoRNA candidates were shown to produce small RNAs between 20–30 nt, 80% of which were associated with ARGONAUT proteins generally, and AGO1b in particular. Overall, our findings provide a comprehensive view of an intermediate-size non-coding transcriptome in a monocot species, which will serve as a useful platform for an in-depth analysis of ncRNA functions.
PMCID: PMC3716300  PMID: 22986792
intermediate-size non-coding RNA;; small nucleolar RNA; rice.
23.  The Translational Apparatus of Plastids and Its Role in Plant Development 
Molecular Plant  2014;7(7):1105-1120.
Systematic reverse genetic approaches in the nuclear and chloroplast genomes have greatly increased our knowledge about the structure, function, and biogenesis of chloroplast ribosomes, and about the molecular mechanisms of plastid protein biosynthesis. They also provided new insights into the regulation of plant development by the activity of plastid gene expression. We review our current knowledge about the translational apparatus of plastids and the impact of plastid translation on plant anatomy and plant morphology.
Chloroplasts (plastids) possess a genome and their own machinery to express it. Translation in plastids occurs on bacterial-type 70S ribosomes utilizing a set of tRNAs that is entirely encoded in the plastid genome. In recent years, the components of the chloroplast translational apparatus have been intensely studied by proteomic approaches and by reverse genetics in the model systems tobacco (plastid-encoded components) and Arabidopsis (nucleus-encoded components). This work has provided important new insights into the structure, function, and biogenesis of chloroplast ribosomes, and also has shed fresh light on the molecular mechanisms of the translation process in plastids. In addition, mutants affected in plastid translation have yielded strong genetic evidence for chloroplast genes and gene products influencing plant development at various levels, presumably via retrograde signaling pathway(s). In this review, we describe recent progress with the functional analysis of components of the chloroplast translational machinery and discuss the currently available evidence that supports a significant impact of plastid translational activity on plant anatomy and morphology.
PMCID: PMC4086613  PMID: 24589494
plastid; translation; ribosome; ribosomal protein; evolution; plastid transformation; retrograde signaling; leaf development; palisade cell.
24.  Aequorin-Based Luminescence Imaging Reveals Stimulus- and Tissue-Specific Ca2+ Dynamics in Arabidopsis Plants 
Molecular Plant  2013;6(2):444-455.
An Aequorin-based Film Adhesive Seedling (FAS) Ca2+ recording system was developed for monitoring Ca2+ in response to various stimuli in Arabidopsis. This system revealed stimulus- and tissue-specific Ca2+ signatures in seedlings with a simple, sensitive, and robust Ca2+ recording.
Calcium ion is a versatile second messenger for diverse cell signaling in response to developmental and environmental cues. The specificity of Ca2+-mediated signaling is defined by stimulus-elicited Ca2+ signature and downstream decoding processes. Here, an Aequorin-based luminescence recording system was developed for monitoring Ca2+ in response to various stimuli in Arabidopsis. With the simple, highly sensitive, and robust Ca2+ recording, this system revealed stimulus- and tissue-specific Ca2+ signatures in seedlings. Cellular Ca2+ dynamics and relationship to Aequorin-based Ca2+ recording were explored using a GFP-based Ca2+ indicator, which suggested that a synchronous cellular Ca2+ signal is responsible for cold-induced Ca2+ response in seedlings, whereas asynchronous Ca2+ oscillation contributes to osmotic stress-induced Ca2+ increase in seedlings. The optimized recording system would be a powerful tool for the identification and characterization of novel components in Ca2+-mediated stress-signaling pathways.
PMCID: PMC3603005  PMID: 23371933
Aequorin; Case12; abiotic stress; calcium; Arabidopsis.
25.  Epigenetic Suppression of T-DNA Insertion Mutants in Arabidopsis  
Molecular Plant  2012;6(2):539-545.
T-DNA insertion mutants have been widely used to define gene functions in Arabidopsis and in other plants. Here, we report an unexpected phenomenon of epigenetic suppression of T-DNA insertion mutants in Arabidopsis. When the two T-DNA insertion mutants, yuc1-1 and ag-TD, were crossed together, the defects in all of the ag-TD plants in the F2 population were partially suppressed regardless of the presence of yuc1-1. Conversion of ag-TD to the suppressed ag-TD (named as ag-TD*) did not follow the laws of Mendelian genetics. The ag-TD* could be stably transmitted for many generations without reverting to ag-TD, and ag-TD* had the capacity to convert ag-TD to ag-TD*. We show that epigenetic suppression of T-DNA mutants is not a rare event, but certain structural features in the T-DNA mutants are needed in order for the suppression to take place. The suppressed T-DNA mutants we observed were all intronic T-DNA mutants and the T-DNA fragments in both the trigger T-DNA as well as in the suppressed T-DNA shared stretches of identical sequences. We demonstrate that the suppression of intronic T-DNA mutants is mediated by trans-interactions between two T-DNA insertions. This work shows that caution is needed when intronic T-DNA mutants are used.
PMCID: PMC3716301  PMID: 22973063
epigenetics; T-DNA mutant; genetic suppression; trans-interaction; YUC.

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