Noncoding RNAs in regulation of plant secondary metabolism.

Plant secondary metabolites (PSMs) are a large class of structurally diverse molecules, mainly consisting of terpenoids, phenolic compounds, and nitrogen-containing compounds, which play active roles in plant development and stress responses. The biosynthetic processes of PSMs are governed by a sophisticated regulatory network at multiple levels. Noncoding RNAs (ncRNAs) such as microRNAs (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs) may serve as post-transcriptional regulators for plant secondary metabolism through acting on genes encoding either transcription factors or participating enzymes in relevant metabolic pathways. High-throughput sequencing technologies have facilitated the large-scale identifications of ncRNAs potentially involved in plant secondary metabolism in model plant species as well as certain species with enriched production of specific types of PSMs. Moreover, a series of miRNA-target modules have been functionally characterized to be responsible for regulating PSM biosynthesis and accumulation in plants under abiotic or biotic stresses. In this review, we will provide an overview of current findings on the ncRNA-mediated regulation of plant secondary metabolism with special attention to its participation in plant stress responses, and discuss possible issues to be addressed in future fundamental research and breeding practice.

Artificial chromosome technology and its potential application in plants.

Plant genetic engineering and transgenic technology are powerful ways to study the function of genes and improve crop yield and quality in the past few years. However, only a few genes could be transformed by most available genetic engineering and transgenic technologies, so changes still need to be made to meet the demands for high throughput studies, such as investigating the whole genetic pathway of crop traits and avoiding undesirable genes simultaneously in the next generation. Plant artificial chromosome (PAC) technology provides a carrier which allows us to assemble multiple and specific genes to produce a variety of products by minichromosome. However, PAC technology also have limitations that may hinder its further development and application. In this review, we will introduce the current state of PACs technology from PACs formation, factors on PACs formation, problems and potential solutions of PACs and exogenous gene(s) integration.

Potential application of TurboID-based proximity labeling in studying the protein interaction network in plant response to abiotic stress.

Abiotic stresses are major environmental conditions that reduce plant growth, productivity and quality. Protein-protein interaction (PPI) approaches can be used to screen stress-responsive proteins and reveal the mechanisms of protein response to various abiotic stresses. Biotin-based proximity labeling (PL) is a recently developed technique to label proximal proteins of a target protein. TurboID, a biotin ligase produced by directed evolution, has the advantages of non-toxicity, time-saving and high catalytic efficiency compared to other classic protein-labeling enzymes. TurboID-based PL has been successfully applied in animal, microorganism and plant systems, particularly to screen transient or weak protein interactions, and detect spatially or temporally restricted local proteomes in living cells. This review concludes classic PPI approaches in plant response to abiotic stresses and their limitations for identifying complex network of regulatory proteins of plant abiotic stresses, and introduces the working mechanism of TurboID-based PL, as well as its feasibility and advantages in plant abiotic stress research. We hope the information summarized in this article can serve as technical references for further understanding the regulation of plant adaptation to abiotic stress at the protein level.

SlMIR164A regulates fruit ripening and quality by controlling SlNAM2 and SlNAM3 in tomato.

MiRNAs are important posttranscriptional regulators of plant development. Many miRNAs, such as the conserved miR164 species, are encoded by families of MIRNA genes, but the specific roles of individual MIRNA genes are largely undefined. Here, we characterize the functions and regulatory mechanisms of SlMIR164A, one of the primary genes of Sly-miR164, in tomato. We show that SlMIR164A is preferentially expressed at late stages of fruit development and plays a vital role in controlling fruit ripening and quality. Loss of function of SlMIR164A by CRISPR/Cas9-mediated mutagenesis results in accelerated fruit ripening and enhanced chloroplast development, which leads to altered sugar and organic acid contents and affects the nutritional quality of fruits. We also show that SlMIR164A modulates fruit ripening and quality through specific target genes, SlNAM2 and SlNAM3, which control key regulators of chloroplast function and fruit ripening processes. MIR164 genes have been shown to play conserved roles in regulating organ ageing, such as leaf senescence and fruit ripening, in a variety of plants, but whether and how their family members in tomato exert the same function remain to be elucidated. Our results reveal a previously undiscovered role of SlMIR164A in ripening control, which will further our understanding of the actions of MIR164 family, as well as the mechanisms of fruit ripening and quality control in tomato. Moreover, as loss of SlMIR164A exhibits minor impacts on organ morphology, our results can be leveraged in tomato breeding for specific manipulation of fruit ripening and quality to facilitate tomato improvement in agriculture.

Sly-miR159 regulates fruit morphology by modulating GA biosynthesis in tomato.

Fruit morphology is an important agronomical trait of many crops. Here, we identify Sly-miR159 as an important regulator of fruit morphology in tomato, a model species of fleshy-fruit development. We show that Sly-miR159 functions through its target SlGAMYB2 to control fruit growth. Suppression of Sly-miR159 and overexpression of SlGAMYB2 result in larger fruits with a reduced length/width ratio, while loss of function of SlGAMYB2 leads to the formation of smaller and more elongated fruits. Gibberellin (GA) is a major phytohormone that regulates fruit development in tomato. We show the Sly-miR159-SlGAMYB2 pathway controls fruit morphology by modulating GA biosynthesis. In particular, we demonstrate that Sly-miR159 promotes GA biosynthesis largely through the direct repression of the GA biosynthetic gene SlGA3ox2 by SlGAMYB2. Together, our findings reveal the action of Sly-miR159 on GA biosynthesis as a previously unidentified mechanism that controls fruit morphology in tomato. Modulating this pathway may have potential applications in tomato breeding for manipulating fruit growth and facilitating the process of fruit improvement.

Noncoding-RNA-Mediated Regulation in Response to Macronutrient Stress in Plants.

Macronutrient elements including nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sulfur (S) are required in relatively large and steady amounts for plant growth and development. Deficient or excessive supply of macronutrients from external environments may trigger a series of plant responses at phenotypic and molecular levels during the entire life cycle. Among the intertwined molecular networks underlying plant responses to macronutrient stress, noncoding RNAs (ncRNAs), mainly microRNAs (miRNAs) and long ncRNAs (lncRNAs), may serve as pivotal regulators for the coordination between nutrient supply and plant demand, while the responsive ncRNA-target module and the interactive mechanism vary among elements and species. Towards a comprehensive identification and functional characterization of nutrient-responsive ncRNAs and their downstream molecules, high-throughput sequencing has produced massive omics data for comparative expression profiling as a first step. In this review, we highlight the recent findings of ncRNA-mediated regulation in response to macronutrient stress, with special emphasis on the large-scale sequencing efforts for screening out candidate nutrient-responsive ncRNAs in plants, and discuss potential improvements in theoretical study to provide better guidance for crop breeding practices.

The pattern of DNA methylation alteration, and its association with the changes of gene expression and alternative splicing during phosphate starvation in tomato.

DNA methylation is changed and associates with gene expression alterations in plant response to phosphate starvation (Pi-), a common stress that impacts plant growth and productivity. However, in the horticultural model species Solanum lycopersicum (tomato), the dynamics of DNA methylation and its relationship with changes in gene transcription and alternative splicing (AS) under Pi- are unknown. Here, we performed integrative methylome and transcriptome analyses of tomato seedlings under Pi-deficient and -sufficient conditions. We found Pi- caused a slight increase in the overall methylation level, with millions of differentially methylated cytosines (DmCs) and a few hundred differentially methylated regions (DMRs). We also identified thousands of differentially expressed (DE) and differential AS (DAS) genes induced by Pi-, and found that DmCs were more abundant in non-expressed genes than in DE or DAS genes. Moreover, DNA methylation alterations weakly correlated with transcription changes but not with DAS events, and hyper-CHH-DMRs overlapping with transposable elements (TEs) were enriched in a subset of Pi starvation response (PSR) genes. We propose that changes in DNA methylation may be associated with the differential expression of some PSR genes, but that most of these changes probably control the expression of nearby TEs, rather than directly affecting the transcription or AS of PSR genes. Besides, the pattern of methylation changes upon Pi- may largely be shaped by TE distributions. Together, our study provides comprehensive insights into the association of DNA methylation with gene transcription and AS under Pi- in tomato and may contribute to unveiling novel roles of epigenetic mechanisms in plant stress response.

Chromosome-scale assembly of the Dendrobium chrysotoxum genome enhances the understanding of orchid evolution.

As one of the largest families of angiosperms, the Orchidaceae family is diverse. Dendrobium represents the second largest genus of the Orchidaceae. However, an assembled high-quality genome of species in this genus is lacking. Here, we report a chromosome-scale reference genome of Dendrobium chrysotoxum, an important ornamental and medicinal orchid species. The assembled genome size of D. chrysotoxum was 1.37 Gb, with a contig N50 value of 1.54 Mb. Of the sequences, 95.75% were anchored to 19 pseudochromosomes. There were 30,044 genes predicted in the D. chrysotoxum genome. Two whole-genome polyploidization events occurred in D. chrysotoxum. In terms of the second event, whole-genome duplication (WGD) was also found to have occurred in other Orchidaceae members, which diverged mainly via gene loss immediately after the WGD event occurred; the first duplication was found to have occurred in most monocots (tau event). We identified sugar transporter (SWEET) gene family expansion, which might be related to the abundant medicinal compounds and fleshy stems of D. chrysotoxum. MADS-box genes were identified in D. chrysotoxum, as well as members of TPS and Hsp90 gene families, which are associated with resistance, which may contribute to the adaptive evolution of orchids. We also investigated the interplay among carotenoid, ABA, and ethylene biosynthesis in D. chrysotoxum to elucidate the regulatory mechanisms of the short flowering period of orchids with yellow flowers. The reference D. chrysotoxum genome will provide important insights for further research on medicinal active ingredients and breeding and enhances the understanding of orchid evolution.

Do Epigenetic Timers Control Petal Development?

Epigenetic modifications include histone modifications and DNA methylation; such modifications can induce heritable changes in gene expression by altering DNA accessibility and chromatin structure. A number of studies have demonstrated that epigenetic factors regulate plant developmental timing in response to environmental changes. However, we still have an incomplete picture of how epigenetic factors can regulate developmental events such as organogenesis. The small number of cell types and the relatively simple developmental progression required to form the Arabidopsis petal makes it a good model to investigate the molecular mechanisms driving plant organogenesis. In this minireview, we summarize recent studies demonstrating the epigenetic control of gene expression during various developmental transitions, and how such regulatory mechanisms can potentially act in petal growth and differentiation.

Metabolite alteration in response to low phosphorus stress in developing tomato fruits.

Alteration of fruit quality caused by environmental stress is a common but largely unresolved issue for plant cultivation and breeding practices. Phosphorus (P) deficiency may interfere with a variety of metabolic processes whose intermediate products are correlated with important fruit quality traits. However, how low P stress affects fruit quality has not been investigated in detail. In this study, we assessed the contents of major metabolites associated with tomato fruit quality under two low P treatments that started at the seedling or flowering stage. The major pigments and the key organic acids related to fruit sourness were differentially over-accumulated as fruit ripened under two low P treatments compared to those under the control treatment, while the total content of soluble sugars contributing to fruit sweetness was substantially reduced under both treatments. These changes were largely attributed to the alteration of enzyme activities in the relevant metabolic pathways. In particular, we found that low P stress from different developmental stages had differential effects on the activation of γ-aminobutyric acid shunt that were likely responsible for the preferential accumulation of different organic acids in tomato fruits. Our study suggested that low P stress strongly affected tomato fruit quality and the effects appeared to be variable under different regimes of low P conditions.

Plant U-box E3 ligases PUB25 and PUB26 control organ growth in Arabidopsis.

Plant organs often grow into a genetically determined size and shape. How organ growth is finely regulated to achieve a well defined pattern is a fascinating, but largely unresolved, question in plant research. We utilised the Arabidopsis petal to study the genetic control of plant organ growth, and identify two closely related U-box E3 ligases PUB25 and PUB26 as important growth regulators by screening the targets of the petal-specific growth-promoting transcription factor RABBIT EARS (RBE). We showed that PUB25 is directly controlled by RBE in petal development in a spatial- and temporal-specific manner and acts as a major target to mediate RBE's function in petal growth. We also showed that PUB25 and PUB26 repress petal growth by restricting the period of cell proliferation, and their regulation appears to be independent of other plant E3 ligase genes implicated in growth control. PUB25 and PUB26 are among the first U-box E3 ligases shown to function in plant growth control. Furthermore, as they were also found to play a vital role in plant stress responses, PUB25 and PUB26 may act as a key hub to integrate developmental and environmental signals for balancing growth and defence in plants.

TCP5 controls leaf margin development by regulating KNOX and BEL-like transcription factors in Arabidopsis.

Development of leaf margins is an important process in leaf morphogenesis. CIN-clade TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PCF) transcription factors are known to have redundant roles in specifying leaf margins, but the specific mechanisms through which individual TCP genes function remain elusive. In this study, we report that the CIN-TCP gene TCP5 is involved in repressing the initiation and outgrowth of leaf serrations by activating two key regulators of margin development, the Class II KNOX factor KNAT3 and BEL-like SAW1. Specifically, TCP5 directly promotes the transcription of KNAT3 and indirectly activates the expression of SAW1. We also show that TCP5 regulates KNAT3 and SAW1 in a temporal- and spatial- specific manner that is largely in accordance with the progress of formation of serrations. This regulation might serve as a key mechanism in patterning margin morphogenesis and in sculpting the final form of the leaf.

Reprogramming of Stem Cell Activity to Convert Thorns into Branches.

Thorns arise from axillary shoot apical meristems that proliferate for a time and then terminally differentiate into a sharp tip. Like other meristems, thorn meristems contain stem cells but, in the case of thorns, these stem cells undergo a programmed cessation of proliferative activity. Using Citrus, we characterize a gene network necessary for thorn development. We identify two Citrus genes, THORN IDENTITY1 (TI1) and THORN IDENTITY2 (TI2), encoding TCP transcription factors, as necessary for stem cell quiescence and thorn identity. Disruption of TI1 and TI2 function results in reactivation of stem cells and concomitant conversion of thorns to branches. Expression of WUSCHEL (WUS) defines the shoot stem cell niche in the apical meristems of many angiosperm species; we show that TI1 binds to the Citrus WUS promoter and negatively regulates its expression to terminate stem cell proliferation. We propose that shifts in the timing and function of components of this gene network can account for the evolution of Citrus thorn identity. Modulating this pathway can significantly alter plant architecture and could be leveraged to improve crop yields.

Unravelling the MicroRNA-Mediated Gene Regulation in Developing Pongamia Seeds by High-Throughput Small RNA Profiling.

Pongamia (Millettia pinnata syn. Pongamia pinnata) is a multipurpose biofuel tree which can withstand a variety of abiotic stresses. Commercial applications of Pongamia trees may substantially benefit from improvements in their oil-seed productivity, which is governed by complex regulatory mechanisms underlying seed development. MicroRNAs (miRNAs) are important molecular regulators of plant development, while relatively little is known about their roles in seed development, especially for woody plants. In this study, we identified 236 conserved miRNAs within 49 families and 143 novel miRNAs via deep sequencing of Pongamia seeds sampled at three developmental phases. For these miRNAs, 1327 target genes were computationally predicted. Furthermore, 115 differentially expressed miRNAs (DEmiRs) between successive developmental phases were sorted out. The DEmiR-targeted genes were preferentially enriched in the functional categories associated with DNA damage repair and photosynthesis. The combined analyses of expression profiles for DEmiRs and functional annotations for their target genes revealed the involvements of both conserved and novel miRNA-target modules in Pongamia seed development. Quantitative Real-Time PCR validated the expression changes of 15 DEmiRs as well as the opposite expression changes of six targets. These results provide valuable miRNA candidates for further functional characterization and breeding practice in Pongamia and other oilseed plants.

Correction: Yu, H.; et al. Molecular Mechanisms of Floral Boundary Formation in Arabidopsis. Int. J. Mol. Sci. 2016, 17, 317.

The authors wish to make the following correction to their paper [1].[...].

Small but powerful: function of microRNAs in plant development.

MicroRNAs (miRNAs) are a group of endogenous noncoding small RNAs frequently 21 nucleotides long. miRNAs act as negative regulators of their target genes through sequence-specific mRNA cleavage, translational repression, or chromatin modifications. Alterations of the expression of a miRNA or its targets often result in a variety of morphological and physiological abnormalities, suggesting the strong impact of miRNAs on plant development. Here, we review the recent advances on the functional studies of plant miRNAs. We will summarize the regulatory networks of miRNAs in a series of developmental processes, including meristem development, establishment of lateral organ polarity and boundaries, vegetative and reproductive organ growth, etc. We will also conclude the conserved and species-specific roles of plant miRNAs in evolution and discuss the strategies for further elucidating the functional mechanisms of miRNAs during plant development.

Comparative Proteomics Analyses of Pollination Response in Endangered Orchid Species Dendrobium Chrysanthum.

Pollination is a crucial stage in plant reproductive process. The self-compatibility (SC) and self-incompatibility (SI) mechanisms determined the plant genetic diversity and species survival. D. chrysanthum is a highly valued ornamental and traditional herbal orchid in Asia but has been declared endangered. The sexual reproduction in D. chrysanthum relies on the compatibility of pollination. To provide a better understanding of the mechanism of pollination, the differentially expressed proteins (DEP) between the self-pollination (SP) and cross-pollination (CP) pistil of D. chrysanthum were investigated using proteomic approaches-two-dimensional electrophoresis (2-DE) coupled with tandem mass spectrometry technique. A total of 54 DEP spots were identified in the two-dimensional electrophoresis (2-DE) maps between the SP and CP. Gene ontology analysis revealed an array of proteins belonging to following different functional categories: metabolic process (8.94%), response to stimulus (5.69%), biosynthetic process (4.07%), protein folding (3.25%) and transport (3.25%). Identification of these DEPs at the early response stage of pollination will hopefully provide new insights in the mechanism of pollination response and help for the conservation of the orchid species.

RABBIT EARS regulates the transcription of TCP4 during petal development in Arabidopsis.

Plant organ growth requires the proper transition from cell proliferation to cell expansion and differentiation. The CIN-TCP transcription factor gene TCP4 and its post-transcriptional regulator microRNA319 play a pivotal role in this process. In this study, we identified a pathway in which the product of the C2H2 zinc finger gene RABBIT EARS (RBE) regulates the transcription of TCP4 during Arabidopsis (Arabidopsis thaliana) petal development. RBE directly represses TCP4 during the early stages of petal development; this contributes to the role of RBE in controlling the growth of petal primordia. We also found that the rbe-1 mutant strongly enhanced the petal phenotypes of tcp4soj6 and mir319a, two mutants with compromised miR319 regulation of TCP4 Our results show that transcriptional and post-transcriptional regulation function together to pattern the spatial and temporal expression of TCP4 This in turn controls petal size and shape in Arabidopsis.