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Increased exposure to environmental stresses due to climate change have adversely affected plant growth and productivity. Upon stress, plants activate a signaling cascade, involving multiple molecules like H 2 O 2, and plant hormones such as salicylic acid (SA) leading to resistance or stress adaptation. However, the temporal ordering and composition of the resulting cascade remains largely unknown. In this study we developed a nanosensor for SA and multiplexed it with H 2 O 2 nanosensor for simultaneous monitoring of stress-induced H 2 O 2 and SA signals when Brassica rapa subsp. Chinensis (Pak choi) plants were subjected to distinct stress treatments, namely light, heat, pathogen stress and mechanical wounding. Nanosensors reported distinct dynamics and temporal wave characteristics of H 2 O 2 and SA generation for each stress. Based on these temporal insights, we have formulated a biochemical kinetic model that suggests the early H 2 O 2 waveform encodes information specific to each stress type. These results demonstrate that sensor multiplexing can reveal stress signaling mechanisms in plants, aiding in developing climate-resilient crops and pre-symptomatic stress diagnoses., (© 2024. The Author(s).)

Nitrogen is an essential macronutrient that is absorbed by roots and stored in leaves, mainly as ribulose-1,5-bisphosphate carboxylase/oxygenase 1,2 . During nitrogen deficiency (-N), plants activate leaf senescence for source-to-sink nitrogen remobilization for adaptative growth 3-6 . However, how -N signals perceived by roots are propagated to shoots remains underexplored. We found that ELF18-INDUCED LONG NONCODING RNA 1 (ELENA1) is -N inducible and attenuates -N-induced leaf senescence in Arabidopsis. Analysis of plants expressing the ELENA1 promoter β-glucuronidase fusion gene showed that ELENA1 is transcribed specifically in roots under -N. Reciprocal grafting of the wild type and elena1 demonstrated that ELENA1 functions systemically. ELENA1 dissociates the MEDIATOR SUBUNIT 19a-ORESARA1 transcriptional complex, thereby calibrating senescence progression. Our observations establish the systemic regulation of leaf senescence by a root-derived long non-coding RNA under -N in Arabidopsis., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Gibberellins (GAs) are a class of phytohormones, important for plant growth, and very difficult to distinguish because of their similarity in chemical structures. Herein, we develop the first nanosensors for GAs by designing and engineering polymer-wrapped single-walled carbon nanotubes (SWNTs) with unique corona phases that selectively bind to bioactive GAs, GA 3 and GA 4 , triggering near-infrared (NIR) fluorescence intensity changes. Using a new coupled Raman/NIR fluorimeter that enables self-referencing of nanosensor NIR fluorescence with its Raman G-band, we demonstrated detection of cellular GA in Arabidopsis , lettuce, and basil roots. The nanosensors reported increased endogenous GA levels in transgenic Arabidopsis mutants that overexpress GA and in emerging lateral roots. Our approach allows rapid spatiotemporal detection of GA across species. The reversible sensor captured the decreasing GA levels in salt-treated lettuce roots, which correlated remarkably with fresh weight changes. This work demonstrates the potential for nanosensors to solve longstanding problems in plant biotechnology.

Phosphate (Pi) availability is a major factor limiting plant growth and development. The key transcription factor controlling Pi-starvation response (PSR) is PHOSPHATE STARVATION RESPONSE 1 (PHR1) whose transcript levels do not change with changes in Pi levels. However, how PHR1 stability is regulated at the post-translational level is relatively unexplored in Arabidopsis thaliana. Inositol polyphosphates (InsPn) are important signal molecules that promote the association of stand-alone SPX domain proteins with PHR1 to regulate PSR. Here, we show that NITROGEN LIMITATION ADAPTATION (NLA) E3 ligase can associate with PHR1 through its conserved SPX domain and polyubiquitinate PHR1 in vitro. The association with PHR1 and its ubiquitination is enhanced by InsP6 but not by InsP5. Analysis of InsPn-related mutants and an overexpression plant shows PHR1 levels are more stable in itpk4-1 and vih2-4/VIH1 amiRNA but less stable in ITPK4 overexpression plants. Under Pi-deficient conditions, nla seedlings contain high PHR1 levels, display long root hair and accumulate anthocyanin in shoots phenocopying PHR1 overexpression plants. By contrast, NLA overexpression plants phenocopy phr1 whose phenotypes are opposite to those of nla. Our results suggest NLA functions as a negative regulator of Pi response by modulating PHR1 stability and the NLA/PHR1 association depends on InsPn levels., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Salicylic acid (SA), a defense hormone produced after pathogen challenge, is critical for plant immunity. Arabidopsis NONEXPRESSER OF PR GENES 1 (NPR1) and its paralogs NPR3 and NPR4 can bind SA and mediate SA signal transduction. NPR1 functions as a transcriptional co-activator to promote defense gene expression, whereas NPR3 and NPR4 have been shown to function as negative regulators in the SA signaling pathway. Although the mechanism about NPR1 regulation has been well studied, how NPR3/NPR4 proteins are regulated in immune responses remains largely unknown. Here, we show that the stability of NPR3/NPR4 is enhanced by SA. In the absence of pathogen challenge, NPR3/NPR4 are unstable and degraded by the 26S proteasome, whereas the increase in cellular SA levels upon pathogen infection suppresses NPR3/NPR4 degradation. We found that UBP12 and UBP13, two homologous deubiquitinases from a ubiquitin-specific protease subfamily, negatively regulate plant immunity by promoting NPR3/NPR4 stability. Our genetic results further showed that UBP12/UBP13-mediated immunity suppression is partially dependent on NPR3/NPR4 functions. By interacting with NPR3 in the nucleus in an SA-dependent manner, UBP12 and UBP13 remove ubiquitin from polyubiquitinated NPR3 to protect it from being degraded. The stabilization of NPR3/NPR4 promoted by UBP12/UBP13 is essential for negative regulation of basal and SA-induced immunity., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

New systems for agrochemical delivery in plants will foster precise agricultural practices and provide new tools to study plants and design crop traits, as standard spray methods suffer from elevated loss and limited access to remote plant tissues. Silk-based microneedles can circumvent these limitations by deploying a known amount of payloads directly in plants' deep tissues. However, plant response to microneedles' application and microneedles' efficacy in deploying physiologically relevant biomolecules are unknown. Here, it is shown that gene expression associated with Arabidopsis thaliana wounding response decreases within 24 h post microneedles' application. Additionally, microinjection of gibberellic acid (GA 3 ) in A. thaliana mutant ft-10 provides a more effective and efficient mean than spray to activate GA 3 pathways, accelerating bolting and inhibiting flower formation. Microneedle efficacy in delivering GA 3 is also observed in several monocot and dicot crop species, i.e., tomato (Solanum lycopersicum), lettuce (Lactuca sativa), spinach (Spinacia oleracea), rice (Oryza Sativa), maize (Zea mays), barley (Hordeum vulgare), and soybean (Glycine max). The wide range of plants that can be successfully targeted with microinjectors opens the doors to their use in plant science and agriculture., (© 2022 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Recent progress in nanotechnology-enabled sensors that can be placed inside of living plants has shown that it is possible to relay and record real-time chemical signaling stimulated by various abiotic and biotic stresses. The mathematical form of the resulting local reactive oxygen species (ROS) wave released upon mechanical perturbation of plant leaves appears to be conserved across a large number of species, and produces a distinct waveform from other stresses including light, heat and pathogen-associated molecular pattern (PAMP)-induced stresses. Herein, we develop a quantitative theory of the local ROS signaling waveform resulting from mechanical stress in planta. We show that nonlinear, autocatalytic production and Fickian diffusion of H 2 O 2 followed by first order decay well describes the spatial and temporal properties of the waveform. The reaction-diffusion system is analyzed in terms of a new approximate solution that we introduce for such problems based on a single term logistic function ansatz. The theory is able to describe experimental ROS waveforms and degradation dynamics such that species-dependent dimensionless wave velocities are revealed, corresponding to subtle changes in higher moments of the waveform through an apparently conserved signaling mechanism overall. This theory has utility in potentially decoding other stress signaling waveforms for light, heat and PAMP-induced stresses that are similarly under investigation. The approximate solution may also find use in applied agricultural sensing, facilitating the connection between measured waveform and plant physiology., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The mediator complex is highly conserved in eukgaryotes and is integral for transcriptional responses. Mediator subunits associate with signal-responsive transcription factors (TF) to activate expression of specific signal-responsive genes. As the key TF of Arabidopsis thaliana senescence, ORESARA1 (ORE1) is required for nitrogen deficiency (-N) induced senescence; however, the mediator subunit that associates with ORE1 remains unknown. Here, we show that Arabidopsis MED19a associates with ORE1 to activate -N senescence-responsive genes. Disordered MED19a forms inducible nuclear condensates under -N that is regulated by decreasing MED19a lysine acetylation. MED19a carboxyl terminus (cMED19a) harbors a mixed-charged intrinsically disordered region (MC-IDR) required for ORE1 interaction and liquid-liquid phase separation (LLPS). Plant and human cMED19 are sufficient to form heterotypic condensates with ORE1. Human cMED19 MC-IDR, but not yeast cMED19 IDR, partially complements med19a suggesting functional conservation in evolutionarily distant eukaryotes. Phylogenetic analysis of eukaryotic cMED19 revealed that the MC-IDR could arise through convergent evolution. Our result of MED19 MC-IDR suggests that plant MED19 is regulated by phase separation during stress responses., (© 2022 Temasek Life Sciences Laboratory. New Phytologist © 2022 New Phytologist Foundation.)

As a key transcription factor in the brassinosteroid (BR) signaling pathway, the activity and expression of BES1 (BRI1-EMS-SUPPRESSOR 1) are stringently regulated. BES1 degradation is mediated by ubiquitin-related 26S proteasomal and autophagy pathways, which attenuate and terminate BR signaling; however, the opposing deubiquitinases (DUBs) are still unknown. Here, we showed that the ubp12-2w/13-3 double mutant phenocopies the BR-deficient dwarf mutant, suggesting that the two DUBs UBP12/UBP13 antagonize ubiquitin-mediated degradation to stabilize BES1. These two DUBs can trim tetraubiquitin with K46 and K63 linkages in vitro. UBP12/BES1 and UBP13/BES1 complexes are localized in both cytosol and nuclei. UBP12/13 can deubiquitinate polyubiquitinated BES1 in vitro and in planta, and UBP12 interacts with and deubiquitinates both inactive, phosphorylated BES1 and active, dephosphorylated BES1 in vivo. UBP12 overexpression in BES1 OE plants significantly enhances cell elongation in hypocotyls and petioles and increases the ratio of leaf length to width compared with BES1 OE or UBP12 OE plants. Hypocotyl elongation and etiolation result from elevated BES1 levels because BES1 degradation is retarded by UBP12 in darkness or in light with BR. Protein degradation inhibitor experiments show that the majority of BES1 can be degraded by either the proteasomal or the autophagy pathway, but a minor BES1 fraction remains pathway specific. In conclusion, UBP12/UBP13 deubiquitinate BES1 to stabilize the latter as a positive regulator for BR responses., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Natural antisense transcripts (NATs) are an important class of non-coding ribonucleic acids (RNAs) that have been shown to regulate gene expression. Using strand-specific RNA sequencing, 36,317 NAT pairs were identified, and 5,536 were specifically expressed under heat stress. We found distinct expression patterns between vegetative and reproductive tissues for both coding genes and genes encoding NATs. Genes for heat-responsive NATs are associated with relatively high levels of H3K4me3 and low levels of H3K27me2/3. On the other hand, small RNAs are significantly enriched in sequence overlapping regions of NAT pairs, and a large number of heat-responsive NATs pairs serve as potential precursors of nat-siRNAs. Collectively, our results suggest epigenetic modifications and small RNAs play important roles in the regulation of NAT expression, and highlight the potential significance of heat-inducible NATs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jin, Ohama, He, Wu and Chua.)

Given the potential applications of gas vesicles (GVs) in multiple fields including antigen-displaying and imaging, heterologous reconstitution of synthetic GVs is an attractive and interesting study that has translational potential. Here, we attempted to express and assemble GV proteins (GVPs) into GVs using the model eukaryotic organism Saccharomyces cerevisiae. We first selected and expressed two core structural proteins, GvpA and GvpC from cyanobacteria Anabaena flos-aquae and Planktothrix rubescens, respectively. We then optimized the protein production conditions and validated GV assembly in the context of GV shapes. We found that when two copies of anaA were integrated into the genome, the chromosomal expression of AnaA resulted in GV production regardless of GvpC expression. Next, we co-expressed chaperone-RFP with the GFP-AnaA to aid the AnaA aggregation. The co-expression of individual chaperones (Hsp42, Sis1, Hsp104, and GvpN) with AnaA led to the formation of larger inclusions and enhanced the sequestration of AnaA into the perivacuolar site. To our knowledge, this represents the first study on reconstitution of GVs in S. cerevisiae. Our results could provide insights into optimizing conditions for heterologous protein production as well as the reconstitution of other synthetic microcompartments in yeast., (© 2021 Wiley-VCH GmbH.)

Changes in light quality caused by the presence of neighbor proximity regulate many growth and development processes of plants. PHYTOCHROME INTERACTING FACTOR 7 (PIF7), whose subcellular localization, DNA-binding properties, and protein abundance are regulated in a photoreversible manner, plays a central role in linking shade light perception and growth responses. How PIF7 activity is regulated during shade avoidance responses has been well studied, and many factors involved in this process have been identified. However, the detailed molecular mechanism by which shade light regulates the PIF7 protein level is still largely unknown. Here, we show that the PIF7 protein level regulation is important for shade-induced growth. Two ubiquitin-specific proteases, UBP12 and UBP13, were identified as positive regulators in shade avoidance responses by increasing the PIF7 protein level. The ubp12-2w/13 - 3 double mutant displayed significantly impaired sensitivity to shade-induced cell elongation and reproduction acceleration. Our genetic and biochemical analysis showed that UBP12 and UBP13 act downstream of phyB and directly interact with PIF7 to maintain PIF7 stability and abundance through deubiquitination., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

We have developed a rapid Raman spectroscopy-based method for the detection and quantification of early innate immunity responses in Arabidopsis and Choy Sum plants. Arabidopsis plants challenged with flg22 and elf18 elicitors could be differentiated from mock-treated plants by their Raman spectral fingerprints. From the difference Raman spectrum and the value of p at each Raman shift, we derived the Elicitor Response Index (ERI) as a quantitative measure of the response whereby a higher ERI value indicates a more significant elicitor-induced immune response. Among various Raman spectral bands contributing toward the ERI value, the most significant changes were observed in those associated with carotenoids and proteins. To validate these results, we investigated several characterized Arabidopsis pattern-triggered immunity (PTI) mutants. Compared to wild type (WT), positive regulatory mutants had ERI values close to zero, whereas negative regulatory mutants at early time points had higher ERI values. Similar to elicitor treatments, we derived an analogous Infection Response Index (IRI) as a quantitative measure to detect the early PTI response in Arabidopsis and Choy Sum plants infected with bacterial pathogens. The Raman spectral bands contributing toward a high IRI value were largely identical to the ERI Raman spectral bands. Raman spectroscopy is a convenient tool for rapid screening for Arabidopsis PTI mutants and may be suitable for the noninvasive and early diagnosis of pathogen-infected crop plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chung, Singh, Huang, Koyyappurath, Seo, Mao, Diloknawarit, Ram, Sarojam and Chua.)

The mechanism of heat stress response in plants has been studied, focusing on the function of transcription factors (TFs). Generally, TFs recruit coactivators, such as Mediator, are needed to assemble the transcriptional machinery. However, despite the close relationship with TFs, how coactivators are involved in transcriptional regulation under heat stress conditions is largely unclear. We found a severe thermosensitive phenotype of Arabidopsis mutants of MED14 and MED17. Transcriptomic analysis revealed that a quarter of the heat stress (HS)-inducible genes were commonly downregulated in these mutants. Furthermore, chromatin immunoprecipitation assay showed that the recruitment of Mediator by HsfA1s, the master regulators of heat stress response, is an important step for the expression of HS-inducible genes. There was a differential requirement of Mediator among genes; TF genes have a high requirement whereas heat shock proteins (HSPs) have a low requirement. Furthermore, artificial activation of HsfA1d mimicking perturbation of protein homeostasis induced HSP gene expression without MED14 recruitment but not TF gene expression. Considering the essential role of MED14 in Mediator function, other coactivators may play major roles in HSP activation depending on the cellular conditions. Our findings highlight the importance of differential recruitment of Mediator for the precise control of HS responses in plants., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)

Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with little or no protein coding potential. The expanding list of lncRNAs and accumulating evidence of their functions in plants have necessitated the creation of a comprehensive database for lncRNA research. However, currently available plant lncRNA databases have some deficiencies, including the lack of lncRNA data from some model plants, uneven annotation standards, a lack of visualization for expression patterns, and the absence of epigenetic information. To overcome these problems, we upgraded our Plant Long noncoding RNA Database (PLncDB, http://plncdb.tobaccodb.org/), which was based on a uniform annotation pipeline. PLncDB V2.0 currently contains 1 246 372 lncRNAs for 80 plant species based on 13 834 RNA-Seq datasets, integrating lncRNA information from four other resources including EVLncRNAs, RNAcentral and etc. Expression patterns and epigenetic signals can be visualized using multiple tools (JBrowse, eFP Browser and EPexplorer). Targets and regulatory networks for lncRNAs are also provided for function exploration. In addition, PLncDB V2.0 is hierarchical and user-friendly and has five built-in search engines. We believe PLncDB V2.0 is useful for the plant lncRNA community and data mining studies and provides a comprehensive resource for data-driven lncRNA research in plants., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Innovative approaches are urgently required to alleviate the growing pressure on agriculture to meet the rising demand for food. A key challenge for plant biology is to bridge the notable knowledge gap between our detailed understanding of model plants grown under laboratory conditions and the agriculturally important crops cultivated in fields or production facilities. This Perspective highlights the recent development of new analytical tools that are rapid and non-destructive and provide tissue-, cell- or organelle-specific information on living plants in real time, with the potential to extend across multiple species in field applications. We evaluate the utility of engineered plant nanosensors and portable Raman spectroscopy to detect biotic and abiotic stresses, monitor plant hormonal signalling as well as characterize the soil, phytobiome and crop health in a non- or minimally invasive manner. We propose leveraging these tools to bridge the aforementioned fundamental gap with new synthesis and integration of expertise from plant biology, engineering and data science. Lastly, we assess the economic potential and discuss implementation strategies that will ensure the acceptance and successful integration of these modern tools in future farming practices in traditional as well as urban agriculture.

Precision agriculture requires new technologies for rapid diagnosis of plant stresses, such as nutrient deficiency and drought, before the onset of visible symptoms and subsequent yield loss. Here, we demonstrate a portable Raman probe that clips around a leaf for rapid, in vivo spectral analysis of plant metabolites including carotenoids and nitrates. We use the leaf-clip Raman sensor for early diagnosis of nitrogen deficiency of the model plant Arabidopsis thaliana as well as two important vegetable crops, Pak Choi (Brassica rapa chinensis) and Choy Sum (Brassica rapa var. parachinensis). In vivo measurements using the portable leaf-clip Raman sensor under full-light growth conditions were consistent with those obtained with a benchtop Raman spectrometer measurements on leaf-sections under laboratory conditions. The portable leaf-clip Raman sensor offers farmers and plant scientists a new precision agriculture tool for early diagnosis and real-time monitoring of plant stresses in field conditions.

Background: Shade avoidance syndrome (SAS) commonly occurs in plants experiencing vegetative shade, causing morphological and physiological changes that are detrimental to plant health and consequently crop yield. As the effects of SAS on plants are irreversible, early detection of SAS in plants is critical for sustainable agriculture. However, conventional methods to assess SAS are restricted to observing for morphological changes and checking the expression of shade-induced genes after homogenization of plant tissues, which makes it difficult to detect SAS early., Results: Using the model plant Arabidopsis thaliana , we introduced the use of Raman spectroscopy to measure shade-induced changes of metabolites in vivo. Raman spectroscopy detected a decrease in carotenoid contents in leaf blades and petioles of plants with SAS, which were induced by low Red:Far-red light ratio or high density conditions. Moreover, by measuring the carotenoid Raman peaks, we were able to show that the reduction in carotenoid content under shade was mediated by phytochrome signaling. Carotenoid Raman peaks showed more remarkable response to SAS in petioles than leaf blades of plants, which greatly corresponded to their morphological response under shade or high plant density. Most importantly, carotenoid content decreased shortly after shade induction but before the occurrence of visible morphological changes. We demonstrated this finding to be similar in other plant species. Comprehensive testing of Brassica vegetables showed that carotenoid content decreased during SAS, in both shade and high density conditions. Likewise, carotenoid content responded quickly to shade, in a manner similar to Arabidopsis plants., Conclusions: In various plant species tested in this study, quantification of carotenoid Raman peaks correlate to the severity of SAS. Moreover, short-term exposure to shade can induce the carotenoid Raman peaks to decrease. These findings highlight the carotenoid Raman peaks as a biomarker for early diagnosis of SAS in plants., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Sweet basil (Ocimum basilicum) plants produce its characteristic phenylpropene-rich essential oil in specialized structures known as peltate glandular trichomes (PGTs). Eugenol and chavicol are the major phenylpropenes produced by sweet basil varieties whose synthetic pathways are not fully elucidated. Eugenol is derived from coniferyl acetate by a reaction catalysed by eugenol synthase. An acyltransferase is proposed to convert coniferyl alcohol to coniferyl acetate which is the first committed step towards eugenol synthesis. Here, we perform a comparative next-generation transcriptome sequencing of different tissues of sweet basil, namely PGT, leaf, leaf stripped of PGTs (leaf-PGT), and roots, to identify differentially expressed transcripts specific to PGT. From these data, we identified a PGT-enriched BAHD acyltransferase gene ObCAAT1 and functionally characterized it. In vitro coupled reaction of ObCAAT1 with eugenol synthase in the presence of coniferyl alcohol resulted in eugenol production. Analysis of ObCAAT1-RNAi transgenic lines showed decreased levels of eugenol and accumulation of coniferyl alcohol and its derivatives. Coniferyl alcohol acts as a common substrate for phenylpropene and lignin biosynthesis. No differences were found in total lignin content of PGTs and leaves of transgenic lines, indicating that phenylpropene biosynthesis is not coupled to lignification in sweet basil., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Nutrient deficiency alters growth and development of crop plants and compromises yield. Real-time non-invasive monitoring of the nutritional status of crops would allow timely applications of fertilizers to optimize for growth and yield at different times of the plant's life cycle. Here, we used Raman spectroscopy to characterize Arabidopsis and two varieties of leafy vegetable crops under nitrogen sufficient and deficient conditions. We showed that the 1046 cm -1 Raman peak serves as a specific signature of nitrogen status in planta , which can be used for early diagnosis of nitrogen deficiency in plants before onset of any visible symptoms. Our research can be applied toward crop management for sustainable and precision agriculture., (Copyright © 2020 Huang, Singh, Park, Chua, Ram and Park.)

Decoding wound signalling in plants is critical for understanding various aspects of plant sciences, from pest resistance to secondary metabolite and phytohormone biosynthesis. The plant defence responses are known to primarily involve NADPH-oxidase-mediated H 2 O 2 and Ca 2+ signalling pathways, which propagate across long distances through the plant vasculature and tissues. Using non-destructive optical nanosensors, we find that the H 2 O 2 concentration profile post-wounding follows a logistic waveform for six plant species: lettuce (Lactuca sativa), arugula (Eruca sativa), spinach (Spinacia oleracea), strawberry blite (Blitum capitatum), sorrel (Rumex acetosa) and Arabidopsis thaliana, ranked in order of wave speed from 0.44 to 3.10 cm min -1 . The H 2 O 2 wave tracks the concomitant surface potential wave measured electrochemically. We show that the plant RbohD glutamate-receptor-like channels (GLR3.3 and GLR3.6) are all critical to the propagation of the wound-induced H 2 O 2 wave. Our findings highlight the utility of a new type of nanosensor probe that is species-independent and capable of real-time, spatial and temporal biochemical measurements in plants.

Several SQUAMASA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors are involved in plant developmental transition from vegetative to reproductive growth. However, the function of SPL10 in regulating floral transition is largely unknown. It is also not known which Mediator subunit mediates SPL10 transcriptional activity. Here, we used overexpression lines and knockout mutants to examine the role of SPL10 in flowering-time regulation and we investigated possible interactions of SPL10 with several mediator subunits in vitro and in vivo. Plants overexpressing SPL10 showed precocious flowering, whereas the triple loss-of-function mutants of SPL10 and its two homologous genes, SPL2 and SPL11, flowered late compared with wild-type plants. We found that SPL10 interacts with MED25, a subunit of the Mediator complex, which bridges transcription factors and RNA polymerase II to facilitate transcription initiation. Genetic analysis showed that MED25 acts downstream of SPL10 to execute SPL10-regulated floral transition. Furthermore, SPL10 was required for MED25 association with the promoters of two target genes, FUL and LFY. We provide evidence that SPL10 recruits MED25 to the promoters of target genes to regulate flowering time. Our results on the SPL10/MED25 module are relevant to the molecular mechanism of other SPL family members., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Nitrogen deficiency (-N) in plants triggers leaf senescence which is regulated by the transcription factor ORE1. Little is known about post-translational regulation of ORE1 in this process. Here, we show that UBP12/UBP13 (ubiquitin-specific protease 12/13) antagonize the action of NLA (nitrogen limitation adaptation) E3 ligase to maintain ORE1 homeostasis. In vitro pull-down and in vivo co-immunoprecipitation assays demonstrated specific binding between UBP12/UBP13 and ORE1. We further analyzed in various genotypes total Chl content and expression levels of senescence-related genes under -N conditions. We found that UBP12/UBP13 can deubiquitinate polyubiquitinated ORE1 in vitro and increase the stability of ORE1 in vivo in MG132/cycloheximide-chase experiments. Plants overexpressing UBP12/UBP13 display accelerated leaf senescence which is reversed by the ore1 mutation. By contrast, the senescence phenotype of plants overexpressing ORE1 is exacerbated by UBP12/UBP13 overexpression. The expression of senescence-related genes tracks the senescence phenotype. ORE1 protein levels can be elevated by UBP12/UBP13 overexpression but decreased in ubp12-2w/13-3. In conclusion, UBP12/UBP13 deubiquitinate ORE1 to stabilize this transcription factor and promote its activity as a positive regulator for leaf senescence under -N conditions. Our study shows that UBP12/UBP13 counteracts the effect of NLA E3 ligase to accelerate leaf senescence under nitrogen starvation., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Plumeria ( Plumeria rubra ), well known for its brightly colored and fragrant flowers, emits a number of floral volatile organic compounds (VOCs). Plumeria flowers emit a total of 43 VOCs including nine phenylpropanoids/benzenoids, such as 2-phenylethanol (2PE), benzaldehyde, 2-phenylacetaldehyde (PAld), ( E/Z ) - phenylacetaldoxime (PAOx), benzyl nitrile (BN), and 2-phenylnitroethane (PN). To identify genes and pathways involved in the production of the major compound 2PE, we analyzed the plumeria floral transcriptome and found a highly expressed, flower-specific gene encoding a cytochrome P450 family 79D protein (PrCYP79D73), which catalyzed the formation of ( E/Z )-PAOx. Feeding experiments with deuterated phenylalanine or deuterated ( E/Z )-PAOx showed that ( E/Z )-PAOx is an intermediate in the biosynthesis of 2PE, as are two nitrogen-containing volatiles, BN and PN, in plumeria flowers. Crude enzyme extracts from plumeria flowers converted l-phenylalanine to ( E/Z )-PAOx, PAld, 2PE, BN, and PN. The biosynthesis of these compounds increased with addition of PrCYP79D73-enriched microsomes but was blocked by pretreatment with 4-phenylimidazole, an inhibitor of cytochrome P450 enzymes. Moreover, overexpression of PrCYP79D73 in Nicotiana benthamiana resulted in the emission of ( E/Z )-PAOx as well as PAld, 2PE, BN, and PN, all of which were also found among plumeria floral VOCs. Taken together, our results demonstrate that PrCYP79D73 is a crucial player in the biosynthesis of the major floral VOC 2PE and other nitrogen-containing volatiles. These volatiles may be required for plant defense as well as to attract pollinators for the successful reproduction of plumeria., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Plant genetic engineering is an important tool used in current efforts in crop improvement, pharmaceutical product biosynthesis and sustainable agriculture. However, conventional genetic engineering techniques target the nuclear genome, prompting concerns about the proliferation of foreign genes to weedy relatives. Chloroplast transformation does not have this limitation, since the plastid genome is maternally inherited in most plants, motivating the need for organelle-specific and selective nanocarriers. Here, we rationally designed chitosan-complexed single-walled carbon nanotubes, utilizing the lipid exchange envelope penetration mechanism. The single-walled carbon nanotubes selectively deliver plasmid DNA to chloroplasts of different plant species without external biolistic or chemical aid. We demonstrate chloroplast-targeted transgene delivery and transient expression in mature Eruca sativa, Nasturtium officinale, Nicotiana tabacum and Spinacia oleracea plants and in isolated Arabidopsis thaliana mesophyll protoplasts. This nanoparticle-mediated chloroplast transgene delivery tool provides practical advantages over current delivery techniques as a potential transformation method for mature plants to benefit plant bioengineering and biological studies.

Plant immune response is initiated upon the recognition of pathogen-associated molecular patterns such as elf18. Previously, we identified an Arabidopsis ELF18-INDUCED LONG NONCODING RNA 1 (ELENA1), as a positive transcriptional regulator of immune responsive genes. ELENA1 associated with Mediator subunit 19a (MED19a) to enhance enrichment of the complex on PATHOGENESIS-RELATED GENE 1 (PR1) promoter. In vitro and in vivo RNA-protein interaction experiments showed that ELENA1 can also interact with FIBRILLARIN 2 (FIB2). Co-immunoprecipitation and bimolecular fluorescence complementation assay showed that FIB2 directly interacts with MED19a in nucleoplasm and nucleolus. Analysis of fib2 mutant showed that FIB2 functions as a negative transcriptional regulator for immune responsive genes, including PR1. Genetic and biochemical analyses demonstrated that ELENA1 can dissociate the FIB2/MED19a complex and release FIB2 from PR1 promoter to enhance PR1 expression. ELENA1 increases PR1 expression by evicting the repressor (FIB2) from the activator (MED19a). Our findings uncover an additional layer of complexity in the transcriptional regulation of plant immune responsive genes by long noncoding RNA., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Long noncoding RNAs (lncRNAs) play important roles in regulating various biological processes including growth and stress responses in plants. RNA-seq data sets provide a good resource to exploring the noncoding transcriptome and studying their comprehensive interactions with the coding transcriptome. Here, we describe computational procedures for studying plant lncRNAs including long intergenic noncoding RNAs (lincRNAs) and long noncoding natural antisense transcripts (lncNATs). Bioinformatics tools for transcriptome assembly, lncRNA identification, and functional interpretations are included. Finally, we also introduce PLncDB, a user-friendly database that provides comprehensive information of plant lncRNAs for researchers to compare their own data sets to those in public database.

RNA-protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA-protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with the MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) to tag lncRNA. Target RNA is tagged with 6xMS2, and MCP and RNA-binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with Agrobacterium suspensions. RNA-protein interaction in vivo is observed by confocal microscopy.

Long noncoding RNAs (lncRNAs) play important roles in several processes including control of gene expression. These RNAs function through binding to histone-modifying complexes and transcriptional machinery including transcription factor, mediator, and RNA polymerase II. We present methods for the discovery and characterization of lncRNAs. RNA immunoprecipitation (RIP) is a modified version of chromatin immunoprecipitation (ChIP), and it is now generally used in lncRNA study. The method allows for testing of lncRNA-protein interactions in vivo. RIP assay facilitates the identification of consensus sequences of preferred binding site for the RNA-binding protein under study, and identification of the binding sites can provide valuable information on the possible mechanism by which the RNA-binding protein functions.

Recent advances in next-generation sequencing have revealed that majority of the plant genome is transcribed into long noncoding RNA (lncRNA). Many lncRNAs function by interacting with proteins and forming regulatory complexes. RNA-protein interactions are vital in controlling core cellular processes like transcription and translation. Therefore, identifying proteins that interact with lncRNAs is the first step to deciphering lncRNA functions. Here, we describe an RNA-protein pull-down assay, which enables the identification of proteins that interact with an RNA under study. As an example, we describe pull-down of proteins interacting with lncRNA ELENA1, which promotes the enrichment of MED19a on PR1 promoter to activate PR1 expression.

Nitrogen is an important macronutrient in plants and its deficiency induces rapid leaf senescence. Two genes, ORE1 and NITROGEN LIMITATION ADAPTATION (NLA), have been implicated in regulating the senescence process but their relationship is unclear 1,2 . Here, we show that nla and pho2 (also known as ubc24) plants develop rapid leaf senescence under nitrogen-starvation condition, whereas ore1 and nla/ore1 and pho2 (ubc24)/ore1 plants stay green. These results suggest that ORE1 acts downstream of NLA and PHO2 (UBC24). NLA interacts with ORE1 in the nucleus and regulates its stability through polyubiquitination using PHO2 (UBC24) as the E2 conjugase. Our findings identified ORE1 as a downstream target of NLA/PHO2 (UBC24) and showed that post-translational regulation of ORE1 levels determines leaf senescence during nitrogen deficiency.

Vegetable oil is an important renewable resource for dietary consumption for human and livestock, and more recently for biodiesel production. Lipid traits in crops are controlled by multiple quantitative trait loci (QTLs) and each of them has a small effect on lipid traits. So far, there is limited success to increase lipid yield and improve lipid quality in plants. Here, we reported the identification of a homolog of APETALA2 (AP2) transcription factor WRINKLED1 (JcWRI1) from an oleaginous plant Jatropha curcas and characterized its function in Jatropha and Arabidopsis thaliana . Using physical mapping data, we located JcWRI1 in a QTL region specifying high oleate and lipid content in Jatropha . Overexpression of JcWRI1 in Jatropha elevated seed lipid content and increased seed mass. Lipid profile in seeds of over-expression plants showed higher oleate content which will be beneficial to improve biodiesel quality. Overexpression of JcWRI1 activated lipid-related gene expression and JcWRI1 was shown to directly bind to the AW-box of promoters of some of these genes. In conclusion, we were able to increase seed lipid content and improve seed lipid quality in Jatropha by manipulating one key transcription factor JcWRI1.

Dispersed H3K27 trimethylation (H3K27me3) of the AGAMOUS (AG) genomic locus is mediated by CURLY LEAF (CLF), a component of the Polycomb Repressive Complex (PRC) 2. Previous reports have shown that the AG second intron, which confers AG tissue-specific expression, harbors sequences targeted by several positive and negative regulators. Using RACE reverse transcription polymerase chain reaction, we found that the AG intron 2 encodes several noncoding RNAs. RNAi experiment showed that incRNA4 is needed for CLF repressive activity. AG-incRNA4RNAi lines showed increased leaf AG mRNA levels associated with a decrease of H3K27me3 levels; these plants displayed AG overexpression phenotypes. Genetic and biochemical analyses demonstrated that the AG-incRNA4 can associate with CLF to repress AG expression in leaf tissues through H3K27me3-mediated repression and to autoregulate its own expression level. The mechanism of AG-incRNA4-mediated repression may be relevant to investigations on tissue-specific expression of Arabidopsis MADS-box genes., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

By modifying the existing cytosolic RNA visualization tool pioneered by Schönberger, Hammes, and Dresselhaus (2012), we developed a method to visualize nuclear-localized RNA. Our method uses (i) an RNA component that consists of an RNA of interest that is fused to a bacteriophage-derived MS2 sequence; and (ii) GFP fused to MS2 coat protein (MSCP), which binds specifically to MS2 as is also the case in the method for cytosolic RNA visualization. The nuclear localization sequence (NLS) at the C-terminal of MSCP-GFP tethers the probe to the nucleus. To reduce background signals in the nucleus, we replaced the NLS with a nuclear export sequence (NES) that anchors the MSCP-GFP probe in the cytosol. Our nuclear RNA visualization method differs from previous methods in two aspects: (i) We used an NES to reduce nuclear background signal so that the MSCP-GFP probe localizes in the cytosol by default; (ii) We added mCherry as a visual marker in the RNA component to increase its efficient usage in a transient system., (© 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Oil palm (Elaeis guineensis Jacq.) is the leading oil-producing crops and the most important edible oil resource worldwide. DNA markers and genetic linkage maps are essential resources for marker-assisted selection to accelerate genetic improvement. We conducted RAD-seq on an Illumina NextSeq500 to discover genome-wide SNPs, and used the SNPs to construct a linkage map for an oil palm (Tenera) population derived from a cross between a Deli Dura and an AVROS Pisifera. The RAD-seq produced 1,076 million single-end reads across the breeding population containing 155 trees. Mining this dataset detected 510,251 loci. After filtering out loci with low accuracy and more than 20% missing data, 11,394 SNPs were retained. Using these SNPs, in combination with 188 anchor SNPs and 123 microsatellites, we constructed a linkage map containing 10,023 markers covering 16 chromosomes. The map length is 2,938.2 cM with an average marker space of 0.29 cM. The large number of SNPs will supply ample choices of DNA markers in analysing the genetic diversity, population structure and evolution of oil palm. This high-density linkage map will contribute to mapping quantitative trait loci (QTL) for important traits, thus accelerating oil palm genetic improvement.

Background: Oil palm is the most productive oil crop and the efficiency of pollination has a direct impact on the yield of oil. Pollination by wind can occur but maximal pollination is mediated by the weevil E. kamerunicus. These weevils complete their life cycle by feeding on male flowers. Attraction of weevils to oil palm flowers is due to the emission of methylchavicol by both male and female flowers. In search for male flowers, the weevils visit female flowers by accident due to methylchavicol fragrance and deposit pollen. Given the importance of methylchavicol emission on pollination, we performed comparative transcriptome analysis of oil palm flowers and leaves to identify candidate genes involved in methylchavicol production in flowers., Results: RNA sequencing (RNA-Seq) of male open flowers, female open flowers and leaves was performed using Illumina HiSeq 2000 platform. Analysis of the transcriptome data revealed that the transcripts of methylchavicol biosynthesis genes were strongly up-regulated whereas transcripts encoding genes involved in lignin production such as, caffeic acid O-methyltransferase (COMT) and Ferulate-5-hydroxylase (F5H) were found to be suppressed in oil palm flowers. Among the transcripts encoding transcription factors, an EAR-motif-containing R2R3-MYB transcription factor (EgMYB4) was found to be enriched in oil palm flowers. We determined that EgMYB4 can suppress the expression of a monolignol pathway gene, EgCOMT, in vivo by binding to the AC elements present in the promoter region. EgMYB4 was further functionally characterized in sweet basil which also produces phenylpropenes like oil palm. Transgenic sweet basil plants showed significant reduction in lignin content but produced more phenylpropenes., Conclusions: Our results suggest that EgMYB4 possibly restrains lignin biosynthesis in oil palm flowers thus allowing enhanced carbon flux into the phenylpropene pathway. This study augments our understanding of the diverse roles that EAR-motif-containing MYBs play to fine tune the metabolic flux along the various branches of core phenylpropanoid pathway. This will aid in metabolic engineering of plant aromatic compounds.

Circadian rhythms of gene expression are generated by the combinatorial action of transcriptional and translational feedback loops as well as chromatin remodelling events. Recently, long noncoding RNAs (lncRNAs) that are natural antisense transcripts (NATs) to transcripts encoding central oscillator components were proposed as modulators of core clock function in mammals (Per) and fungi (frq/qrf). Although oscillating lncRNAs exist in plants, their functional characterization is at an initial stage. By screening an Arabidopsis thaliana lncRNA custom-made array we identified CDF5 LONG NONCODING RNA (FLORE), a circadian-regulated lncRNA that is a NAT of CDF5. Quantitative real-time RT-PCR confirmed the circadian regulation of FLORE, whereas GUS-staining and flowering time evaluation were used to determine its biological function. FLORE and CDF5 antiphasic expression reflects mutual inhibition in a similar way to frq/qrf. Moreover, whereas the CDF5 protein delays flowering by directly repressing FT transcription, FLORE promotes it by repressing several CDFs (CDF1, CDF3, CDF5) and increasing FT transcript levels, indicating both cis and trans function. We propose that the CDF5/FLORE NAT pair constitutes an additional circadian regulatory module with conserved (mutual inhibition) and unique (function in trans) features, able to fine-tune its own circadian oscillation, and consequently, adjust the onset of flowering to favourable environmental conditions., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

RNA-Protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA/protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-Protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-Protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) ( Schonberger et al. , 2012 ). Target RNA is tagged with 6xMS2 and MCP and RNA binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with Agrobacterium suspensions. RNA-Protein interaction in vivo is observed by confocal microscope., (Copyright © 2017 The Authors; exclusive licensee Bio-protocol LLC.)

The 3' untranslated regions (3' UTRs) of mRNAs play important roles in the regulation of mRNA localization, translation, and stability. Alternative cleavage and polyadenylation (APA) generates mRNAs with different 3' UTRs, but the involvement of this process in stress response has not yet been clarified. Here, we report that a subset of stress-related genes exhibits 3' UTR extensions of their mRNAs during dehydration stress. These extended 3' UTRs have characteristics of long noncoding RNAs and likely do not interact with miRNAs. Functional studies using T-DNA insertion mutants reveal that they can act as antisense transcripts to repress expression levels of sense genes from the opposite strand or can activate the transcription or lead to read-through transcription of their downstream genes. Further analysis suggests that transcripts with 3' UTR extensions have weaker poly(A) signals than those without 3' UTR extensions. Finally, we show that their biogenesis is partially dependent on a trans -acting factor FPA. Taken together, we report that dehydration stress could induce transcript 3' UTR extensions and elucidate a novel function for these stress-induced 3' UTR extensions as long noncoding RNAs in the regulation of their neighboring genes., (© 2017 Sun et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Magnolia champaca, commonly known as champak is a well-known tree due to its highly fragrant flowers. Champak floral scent is attributed to a complex mix of volatile organic compounds (VOCs). These aromatic flowers are widely used in flavors and fragrances industry. Despite its commercial importance, the VOC biosynthesis pathways in these flowers are largely unknown. Here, we combine metabolite and RNA sequencing (RNA-seq) analyses of fully opened champak flowers to discover the active VOC biosynthesis pathways as well as floral scent-related genes., Results: Volatile collection by headspace method and analysis by gas chromatography-mass spectrometry (GC-MS) identified a total of 43 VOCs from fully opened champak flowers, of which 46.9% were terpenoids, 38.9% were volatile esters and 5.2% belonged to phenylpropanoids/benzenoids. Sequencing and de novo assembly of champak flower transcriptome yielded 47,688 non-redundant unigenes. Transcriptome assembly was validated using standard polymerase chain reaction (PCR) based approach for randomly selected unigenes. The detailed profiles of VOCs led to the discovery of pathways and genes involved in floral scent biosynthesis from RNA-seq data. Analysis of expression levels of many floral-scent biosynthesis-related unigenes in flowers and leaves showed that most of them were expressed higher in flowers than in leaf tissues. Moreover, our metabolite-guided transcriptomics, in vitro and in vivo enzyme assays and transgenic studies identified (R)-linalool synthase that is essential for the production of major VOCs of champak flowers, (R)-linalool and linalool oxides., Conclusion: As our study is the first report on transcriptome analysis of Magnolia champaca, this transcriptome dataset that serves as an important public information for functional genomics will not only facilitate better understanding of ecological functions of champak floral VOCs, but also provide biotechnological targets for sustainable production of champak floral scent.