Plant Molecular Biology

Plants must quickly adapt to a changing environment in order to maintain their fitness. One rapid adaptation mechanism that promotes plasticity is epigenetic memory, which may provide long-lived organisms the precious time needed to adapt to climate change. In this study, we used the perennial plant Fragaria vesca as a model to determine how the methylome and transcriptome adapt to elevated temperatures (28 vs. 18 {degrees}C) over three asexual generations. Changes in flowering time, stolon number, and petiole length were induced in responses to temperature treatment in one or more ecotypes after three asexual generations in a manner indicative of an epigenetic memory. Induced methylome changes differed between four different ecotypes from Norway, Iceland, Italy, and Spain, but there were also some shared responses. Elevated temperature conditions induced significant phenotypic and methylation changes, particularly in the Norwegian ecotype. Most of the differentially methylated regions (DMRs) were in the CHG context, and most CHG and CHH DMRs were hypermethylated. Notably, the four ecotypes shared only eight CHG DMR peaks. Several differentially methylated genes (DMGs) also showed a change in gene expression. Ecotype-specific methylation and expression patterns were observed for genes related to gibberellin metabolism, flowering time, epigenetics. Furthermore, when repetitive elements (REs) were found near ({+/-}2 kb) or inside a gene, they showed a negative correlation with gene expression. In conclusion, phenotypic changes induced by elevated temperatures during asexual reproduction were accompanied by changes in DNA methylation patterns. Also, positional influences of REs impacted gene expression, indicating that DNA methylation may be involved in both general and ecotype-specific phenotypic plasticity in F. vesca.

The ethylene response factor (ERF) transcription factor is a subfamily of AP2/ERF superfamily in plants, which plays multiple roles in plant growth and development as well as stress response. In this study, we found that the GsERF gene from BW69 line of wild soybean held a constitutive expression pattern and induced by aluminum stress with more transcripts in soybean root. The putative GsERF protein containing an AP2 domain was in the nucleus and transactivation activity. In addition, the overexpression of the GsERF gene enhanced root relative length rate in Arabidopsis and shallow staining by hematoxylin under the treatments of AlCl3. The ethylene synthesis related genes such as ACS4, ACS5 and ACS6 are upregulated in the GsERF overexpressed plants than those in wild type plants under the treatment of AlCl3. Furthermore, expression levels of stress/ABA-responsive marker genes, including ABI1, ABI2, ABI4, ABI5, RD29B and RD22 in transgenic lines compared with those in wild type Arabidopsis were affected by AlCl3 treatments. Taken together, the results indicate that overexpression of GsERF may enhance aluminum tolerance through an ethylene-mediated pathway and/or ABA signaling pathway in Arabidopsis thaliana.View Full Text

MicroRNAs (miRNAs) negatively regulate gene expression by cleaving the target mRNA and/or impairing its translation, thereby playing a crucial role in plant development and environmental stress responses. In Arabidopsis, MIR840 is located within the overlapping 3UTR of PPR and WHIRLY3 (WHY3), both being predicted targets of miR840. Gain- and loss-of-function of miR840 in Arabidopsis resulted in opposite senescent phenotypes. Highest expression of pri-miR840 is observed at senescence initiation, and is negatively correlated with a significant reduction of PPR transcripts but not of WHY3. Although WHY3 transcript levels were not significantly affected by miR840 overexpression, its protein synthesis was strongly reduced. Mutating the cleavage sites or replacing the target sequences abolishes the miR840-mediated degradation of PPR transcripts and inhibition of WHY3 translation. In support for this, concurrent knock-down of both PPR and WHY3 in the WT resulted in the senescent phenotype resembling that of the miR840-overexpressing mutant. This indicates that both PRR and WHY3 are targets in the miR840-regulated senescent pathway. Moreover, single knockout mutant of PPR or WHY3 shows a convergent up-regulated subset of senescence-associated genes, which are also found among those induced by miR840 overexpression. Our data provide evidences for a regulatory role of miR840 in plant senescence. HighlightMicroRNA840 (miR840) has a unique miRNA-target configuration regulating PPR and WHIRLY3 genes in Arabidopsis. MiR840 is highly expressed at the onset of plant senescent stage. Both PPR and WHIRLY3 transcripts are specifically targeted in vivo within their 3UTR region by mature miR840 or its star strand in vivo. Interestingly, PPR expression is mainly repressed on mRNA transcript level by cleavage, while WHIRLY3 is predominantly translationally inhibited. We conclude that miR840 enhances plant senescence via post transcriptional gene silencing of PPR and WHIRLY3, which appear to be novel negative joint regulators of plant senescence. Footnote: The author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the intructions for Authors is: Ying Miao (ymiao@fafu.edu.cn)

Tomato (Solanum lycopersicum L.) is one of the highest consumable fruit crops, rich in nutrients, and has been an important target for enhancing the accumulation of various metabolites. Tomato also contains cholesterol-derived molecules, steroidal glycoalkaloids (SGAs), which contribute to pathogen defence but are toxic to humans and considered anti-nutritional compounds. Previous studies suggest the role of various transcription factors in SGA biosynthesis; however, the role of light and associated regulatory factors has not been studied in tomatoes. Here, we demonstrated that SGA biosynthesis is regulated by light through the ELONGATED HYPOCOTYL 5 homolog, SlHY5, by binding to light-responsive G-boxes present in the promoters of the structural and regulatory genes. Our analysis suggests that SlHY5 could complement the Arabidopsis thaliana and Nicotiana tabacum, hy5 mutants at molecular, morphological, and biochemical levels. We report the development of CRISPR/Cas9-based knockout mutant plants of tomato, slhy5CR, and show down-regulation of the SGA and phenylpropanoid pathway genes leading to a significant reduction in SGA (-tomatine and dehydrotomatine) and flavonol contents, whereas SlHY5 overexpression (SlHY5OX) plants show opposite effect. An enhanced SGA and flavonol levels in SlHY5OX lines provided tolerance against Alternaria solani fungus, while SlHY5CR was susceptible to the pathogen. This study advances our understanding of the HY5-dependent light-regulated biosynthesis of SGAs and flavonoids and their role in biotic stress in tomatoes. One Sentence SummaryLight-associated transcription factor, ELONGATED HYPOCOTYL 5, regulates biosynthesis of anti-nutrient molecules, steroidal glycoalkaloids, and fungal tolerance in tomato

Plants can not move automatically, thus they have to perceive the changing environment (light, temperate and so on) by developing the adapted phenotypes. Light-induced hypocotyl length is an ideal phenotype for studying how plants response to light. So far, many signaling components in light-induced hypocotyl growth have been reported. Here, we focused on identifying transcription factors (TFs) involved in blue light-induced hypocotyl growth by constructing Arabidopsis TFs overexpressing lines and screening blue light-induced hypocotyl length. Finally, we found that three NF-YC proteins, NF-YC7, NF-YC5 and NF-YC8 (NF-YCs as short name), develop longer hypocotyls than the wild type under blue light. While deficient mutants, nf-yc5nf-yc7 and nf-yc7nf-yc8, fail to promote the hypocotyl elongation under blue light. NF-YCs physically interacted with CRY2 and PIF4/5, while the NF-YCs-PIF4/5 interactions were repressed by CRY2. Moreover, overexpression of CRY2 or deficiency of PIF4/5 repressed the hypocotyl elongation induced by NF-YC7 under blue light. Further investigation revealed that NF-YC7 increased the blue light-induced CRY2 degradation and regulated the activities of PIF4/5. Taking together, this study provided a new insight into that NF-YCs function as CRY2-and PIF4/5-interacting proteins and modulate their stabilization to repress the blue light-mediated hypocotyl growth. Author SummaryLight is an essential environmental factor for plants growth and development. Plants response to light signaling by displaying short hypocotyls, green leaves, and so on. The mechanisms of light responding to light have aroused extensive attention. In this study, we clarified that NF-YC family members NF-YC5/7/8 interact with CRY2, PIF4/5 and modulate their stabilization to repress the blue light-mediated hypocotyl growth. It was the first time for reporting NF-Y family members function as CRY2-and PIF4/5-interacting proteins. Therefore, this study provides a novel understanding how plants adapt to light.

The APETALA2/ethylene response factor (AP2/ERFs) are pivotal in regulating abiotic stress responses in plants. However, the specific role of ERFs in kenafs response to drought stress remains unclear. In this study, a transcription factors HcERF5 was isolated from kenaf and its role in drought stress tolerance was analyzed. HcERF5 was found to localize in both nucleus and cytoplasm and could be significantly induced by polyethylene glycol-6000 (PEG-6000) and abscisic acid (ABA) in kenaf seedlings. In transgenic Arabidopsis expressing the HcERF5 promoter-driven {beta}-glucuronidase (GUS), strong GUS activity was observed in roots, stems, and leaves. Overexpression of HcERF5 in Arabidopsis enhanced seed germination rates under drought or ABA stress and improved drought tolerance in seedlings by increasing antioxidant enzyme activities, whereas aterf5 knockout lines exhibited the opposite trend. Additionally, HcERF5 overexpressing Arabidopsis showed significantly increased drought tolerance and reduced sensitivity to ABA. Furthermore, virus-induced gene silencing (VIGS) of HcERF5 in kenaf reduced drought tolerance, as evidenced by decreased antioxidant enzyme activity, increased stomatal aperture, and elevated levels of malondialdehyde (MDA), reactive oxygen species (ROS), and proline under drought stress. RNA-seq analysis further revealed that HcERF5 directly regulated ABA signaling pathway. Yeast-two-hybrid (Y2H) assays revealed 29 proteins that interact with HcERF5. Among them, the expression of downstream drought stress-related genes HcPRK, HcRD22, HcMAP2, HcCAB, HcCS, and HcCCoAOMT3 were significantly reduced in HcERF5-silenced plants. Overall, this study highlights the significant potential of HcERF5 in enhancing drought tolerance in kenaf. HighlightO_LIHcERF5 overexpression enhanced drought tolerance in kenaf, however silencing increased drought sensitivity. C_LIO_LIHcERF5 regulates ABA synthesis and increases kenaf stomatal conductance and density under drought stress. C_LIO_LIHcERF5 regulates plant hormone signal transduction, MAPK signalling, and phenylpropanoid biosynthesis in kenaf under drought stress. C_LIO_LIProtein interaction revealed HcERF5 interacts with six stress-response genes. C_LI

Mitogen activated protein kinase (MAPK) cascade is evolutionary conserved universal signal transduction module that plays central role in the growth and development of plants as well as in biotic and abiotic stress response. Although, MAPKs have been investigated in several model plants, no systematic analysis has been conducted in jute species (Corchorus olitorius and C. capsularis) even though, their genome sequencing has been completed. In the present study we identified 11 and 12 putative MAPKs in C. olitorius and C. capsularis using their genomic database, respectively. Here we provide a comprehensive bioinformatics analysis of the MAPK family from both Corchorus species including identification and nomenclature, chromosomal localization, sequence alignment, domain and Motif, gene structure, phylogenetic, functional analysis and investigation of expression analysis in response to abiotic stress and fiber cell development. The phylogenetic analysis of predicted MAPKs were clustered into four different clades and assigned with specific name based on their orthology based evolutionary relationship with Arabidopsis. Structural analysis of the MAPK genes revealed that there was a large variation among the exon number in both Corchorus species ranged from 2 to 11 but genes with the same clade had similar exon-intron structure. The sequence alignment analysis concede the presence of several conserved domain and motif including crucial signature phosphorylation motif TDY or TEY where first one is harbor in group D sequence and rest of the sequence contain TEY motif in their activation loop. Transcriptome analysis against salinity, drought along with fiber cell formation showed that MAPK4-1 genes in both jute genome highly expressed and may play a potential role in jute on adverse condition as well as jute fiber formation. These findings yielded new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in jute, that lead to potential applications in the genetic improvement of jute cultivars.

MicroRNAs (miRNAs) regulate gene expression affecting a variety of plant developmental processes. The evolutionary position of Marchantia polymorpha makes it a significant model to understand miRNA-mediated gene regulatory pathways in plants. Previous studies focused on conserved miRNA-target mRNA modules showed their critical role in Marchantia development. Here, we demonstrate that differential expression of conserved miRNAs and their targets in selected organs of Marchantia additionally underlines their role in regulating fundamental developmental processes. The main aim of this study was to characterize selected liverwort-specific miRNAs, as there is a limited knowledge on their biogenesis, accumulation, targets, and function in Marchantia. We demonstrate their differential accumulation in vegetative and generative organs. We reveal that all liverwort-specific miRNAs examined are encoded by independent transcriptional units. MpmiR11737a, MpmiR11887 and MpmiR11796, annotated as being encoded within protein-encoding genes, have their own independent transcription start sites. The analysis of selected liverwort-specific miRNAs and their pri-miRNAs often reveal correlation in their levels, suggesting transcriptional regulation. However, MpmiR11796 shows a reverse correlation to its pri-miRNA level, suggesting post-transcriptional regulation. Moreover, we identify novel targets for selected liverwort-specific miRNAs and demonstrate an inverse correlation between their expression and miRNA accumulation. In the case of one miRNA precursor, we provide evidence that it encodes two functional miRNAs with two independent targets. Overall, our research sheds light on liverwort-specific miRNA gene structure, provides new data on their biogenesis and expression regulation. Furthermore, identifying their targets, we hypothesize the potential role of these miRNAs in early land plant development and functioning.

It is widely known that protein kinases (PKs) play a fundamental role in regulating various metabolic processes in plants, from development to response to the environment. However, a detailed characterization of this superfamily is still lacking for several species, such as cucumber (Cucumis sativus), especially regarding their involvement in the response to Powdery Mildew (PM) caused by Podosphaera xanthii. This study aimed to characterize the cucumber PK family, shedding light on its genomic distribution, classification, and expression patterns triggered by P. xanthii. The hidden Markov models (HMMs) analysis uncovered 835 PKs in the cucumber kinome, distributed across its seven chromosomes, and categorized into 20 distinct groups and 123 families, with the RLK group being the most abundant. Evidence of tandem duplication of PK genes was also observed, enriching our understanding of cucumber PKs. To investigate the expression profiles of PK genes in cucumber, we analyzed the transcription levels of all 835 PK genes in RNA-seq data from leaves of resistant and susceptible cultivars of cucumber to P. xanthii, which were artificially inoculated. Depending on the treatment, DEGs ranged from 319 to 1,690, with PK DEGs ranging from 8 to 105. The number of PK DEGs varied between the different contrasts analyzed. Notably, we observed a greater number of PK DEGs in susceptible genotypes when challenged by the pathogen. Our findings indicate the role of specific cucumber PKs in regulating metabolic processes in the context of plant-pathogen interactions and pave the way for further research into the intricate mechanisms underlying cucumber responses to Powdery Mildew. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/532963v3_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@11340b8org.highwire.dtl.DTLVardef@fe1896org.highwire.dtl.DTLVardef@5de8org.highwire.dtl.DTLVardef@1564d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pearl millet is a significant crop tolerant to abiotic stresses and is a staple food of arid regions. However, its underlying mechanisms of stress tolerance is not fully understood. Plant survival is regulated by the ability to perceive a stress signal and induce appropriate physiological changes. Here, we screened for genes regulating physiological changes such as chlorophyll content (CC) and relative water content (RWC) in response to abiotic stress. Using weighted gene co-expression network analysis (WGCNA) and clustering associated changes in physiological traits, i.e., CC and RWC with gene expression. A group of genes showing correlation with traits was identified as modules, and different color names were used to denote a particular module. In WGCNA, the darkgreen module (7082 genes) showed a significant positive correlation with CC, and the black (1393 genes) module was negatively correlated with CC and RWC. Analysis of the module positively correlated with CC highlighted ribosome synthesis and plant hormone signaling as the most significant pathways. Potassium transporter 8 and monothiol glutaredoxin were reported as the topmost hub genes in the darkgreen module. In Clust analysis, 2987 genes were found to display a correlation with increasing CC and RWC. Further, the pathway analysis of these clusters identified ribosome and thermogenesis as positive regulators of RWC and CC, respectively. Our study provides novel insights into the molecular mechanisms regulating CC and RWC in pearl millet.

Inherited genetic change can happen in flax (Linum usitatissimum) due to environmental stress. The change can result in different phenotypes in flax progeny. The genetic changes can be induced during one generation and can result in stable genotypes in the next generation. Also the genetic changes are precise and repeatable that homozygous individuals show the same genome reorganization at specific sites in their progeny. Therefore, the genetic re-arrangements are not the result of a random process but a preference for a particular DNA structure, which indicates that possible new genes or functional fragments are formed during this process. The genomes of different varieties of flax are compared to explain the detail of the rapid but intentional genomic changes and regions of variation identified by whole genome comparisons. Possible mechanisms and the potential causes of these rearrangements bring a new light to modern plant genome studies and molecular evolution research.

Secondary metabolites produced by the phenylpropanoid pathway, which is regulated by transcription factors of the MYB family, play crucial roles in this early phase of fruit development. The MYB46 transcription factor is a key regulator of secondary cell wall structure and lignin and flavonoid biosynthesis in many plants, but little is known about its activity in flowers and berries in F. vesca. For functional analysis of FvMYB46, we designed a CRISPR-Cas9 construct with an endogenous F. vesca specific U6-promoter for efficient and specific expression of two gRNAs targeting the first exon of FvMYB46. This generated mutants with an in frame 81-bp deletion of the first conserved MYB-domain or an out of frame 82-bp deletion potentially knocking out the gene function. In both types of mutant plants, pollen germination and the frequency of flowers developing to mature berries was significantly reduced compared to wild type. Transcriptomic analysis of flowers demonstrated that FvMYB46 is positively regulating the expression of genes involved in pollen germination, homeostasis of reactive oxygen species (ROS) and the phenylpropanoid pathway, including secondary cell wall biosynthesis and flavonoid biosynthesis, while has a negative impact on carbohydrate metabolism. In FvMYB46-mutant flowers, the flavonols and flavan-3-olscontent, especially epicatechin, quercetin-glucoside and kaempferol-3-coumaroylhexoside were reduced, and we observed a local reduction of lignin content in anthers. Together these results suggest that MYB46 control fertility and efficient fruit set by regulating cell wall structure, flavonoid biosynthesis, carbohydrate metabolism and ROS-signaling in flowers and early fruit development in F. vesca.

MADS-box transcription factors (TFs) have been extensively studied in seed plants, but their functions in non-seed plants remain comparatively underexplored. CsubMADS1, a MADS-box TF from the microalga Coccomyxa subellipsoidea C-169, was previously shown to influence developmental processes and starvation-associated responses. In this study, overexpression of CsubMADS1 was associated with increased tolerance to nutrient-limiting conditions, particularly during stationary phase and nitrogen starvation. Overexpressing lines exhibited reduced neutral lipid droplet formation and decreased mucilage staining under these conditions--physiological traits typically linked to stress-associated responses in microalgae. RNA-seq analysis identified 19 chlorophyll a/b binding protein (CAB) genes as upregulated in the overexpressors, six of which were validated by qPCR. In wild-type cells, CsubMADS1 was upregulated and CAB transcripts were downregulated during nitrogen starvation, indicating that both the transcription factor and these candidate downstream genes respond to nutrient stress. Six CAB genes contained CArG motifs within their 2 kb upstream regions, and CArG boxes from CAB24 and CAB8 were tested for binding. CAB24 belongs to the stress-responsive LI818 family. Agarose-based EMSA provided preliminary evidence of CsubMADS1 interaction with the CAB24 and CAB8 CArG motifs, with the enriched sequence resembling the AGL15-type C(A/T)8G motif. These observations suggest that CAB gene expression is associated with stress responses and that CsubMADS1 may regulate a subset of CABs, although additional assays are required to confirm direct regulation.

In plants, 14-3-3 proteins are recognized as mediators of signal transduction and function in both development and stress response. However, their functions have not been reported in the C4 crop foxtail millet. Here, phylogenetic analysis categorized foxtail millet 14-3-3s (SiGRFs) into ten discrete groups (Clusters I to {square}). Transcriptome and qPCR analyses showed that all the SiGRFs responded to at least one abiotic stress. All but one SiGRF-overexpressing (OE) Arabidopsis thaliana line (SiGRF1) exhibited insensitivity to abiotic stresses during seed germination and seedling growth. Compared with the Col-0 wild-type, SiGRF1-OEs had slightly lower germination rates and smaller leaves. However, flowering time of SiGRF1-OEs occurred earlier than that of Col-0 under high-salt stress. Interaction of SiGRF1 with a foxtail millet E3 ubiquitin-protein ligase (SiRNF1/2) indicates that the proteinase system might hydrolyse SiGRF1. Further investigation showed that SiGRF1 localized in the cytoplasm, and its gene was ubiquitously expressed in various tissues throughout various developmental stages. Additionally, flowering-related genes, WRKY71, FLOWERING LOCUS T, LEAFY and FRUITFULL, in SiGRF1-OEs exhibited considerably higher expression levels than those in Col-0 under salinity-stressed conditions. Results suggest that SiGRF1 hastens flowering, thereby providing a means for foxtail millet to complete its life cycle and avoid further salt stress. HighlightSiGRFs in foxtail millet: SiGRF1 hastens flowering in transgenic Arabidopsis thaliana exposed to salt stress

Respiratory burst oxidase homolog (Rboh), generate reactive oxygen species (ROS) to maintain normal growth and pathogen induced defence responses in plants. In Aquilaria plants, wounding and fungal invasion results in the biosynthesis of secondary metabolites as a defence response which with due course develop into agarwood. During pathogen invasion, Aquilaria tree accumulate ROS species through the action of Rboh enzymes. Although in agarwood formation role of Rboh gene family has been implicated, an comprehensives study on Rboh gene family and information of its role during agarwood formation in missing. In this study, seven Rboh genes were identified from the genomes of two Aquilaria species viz., Aquilaria agallocha and Aquilaria sinensis and phylogenetically classified into five groups. Stress response, hormone regulation, and development related regulatory elements were identified in the promoter regions. The protein sequences comprised of four conserved domains, an EF-hand domain, and a transmembrane region which they probably utilise for MAPK signaling, plant-pathogen interaction and plant hormone signal transduction pathways. Expression analyses revealed that among the seven members, AaRbohA and AaRhobC were involved in generation of ROS species, and also probably play role in agarwood formation. These findings provide valuable information regarding the Rboh members of A. agallocha which can be further used for functional analyses for in-depth understanding of ROS mediated signalling and regulation of agarwood formation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/539809v1_figu1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@169f501org.highwire.dtl.DTLVardef@1541667org.highwire.dtl.DTLVardef@18ad549org.highwire.dtl.DTLVardef@1b8b7b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundUpon exposure to unfavorable environmental conditions, plants need to respond quickly to maintain their homeostasis. For instance, physiological, biochemical and transcriptomical changes must occur during interactions with pathogens, this causing the triggering of pathogen- and plant-derived molecules. In the case of Vanilla planifolia Jacks., a worldwide economically important crop, it is susceptible to Fusarium oxysporum f. sp. vanillae. This pathogen causes root and stem rot in vanilla plants that finally leads to plant death. To further investigate how vanilla plants respond at the transcriptional level upon infection with F.oxysporum f. sp. vanillae, we employed the RNA-Seq approach to analyze the dynamics of whole-transcriptome changes during two-time frames of the infection.\n\nResultsAnalysis of global gene expression profiles indicated that a major transcriptional change occurs at 2 dpi, in comparison to 10 dpi, whereas 3420 genes were found with a differential expression at 2 dpi, only 839 were identified at 10 dpi. The analysis of the transcriptional profile at 2 dpi suggests that vanilla plants prepare to counter the infection by gathering a pool of translational regulation-related transcripts.\n\nConclusionsWe propose that the plant-pathogen interaction at early stages causes a transcriptional reprogramming coupled with a translational regulation. Altogether, this study provides the identification of molecular players that could help to fight the most damaging disease of vanilla, where ribosomal proteins and regulation of the translational mechanism are critical. These are insights into the defense responses of V. planifolia Jacks., providing the basis for the understanding of the plant early response towards biotic stress.

Chlorophyll degradation and carotenoid biosynthesis, which occur almost simultaneously during fruit ripening, are essential for coloration and nutritional value of fruits. However, the synergistic regulation of these two processes at transcriptional level remains largely unknown. Here, we identified a novel WRKY transcription factor CrWRKY42 from the transcriptome data of the yellowish bud mutant Jinlegan tangor (MT) and its wild type Shiranuhi tangor (WT), which was involved in the transcriptional regulation of both chlorophyll degradation and carotenoid biosynthesis pathways. CrWRKY42 activated the expression of {beta}-carotene hydroxylase 1 (CrBCH1) by directly binding to its promoter. Overexpression and interference of CrWRKY42 in citrus calli demonstrated that CrWRKY42 promoted carotenoid accumulation by inducing the expression of multiple carotenoid biosynthetic genes. Further assays confirmed that CrWRKY42 also directly bound to and activated the promoters of the genes involved in the carotenoid biosynthesis, including phytoene desaturase (CrPDS) and lycopene {beta}-cyclase 2 (CrLCYB2). In addition, CrWRKY42 could also bind to the promoter of STAY-GREEN (CrSGR) and activated its expression, thus promoting chlorophyll degradation. Overexpression and silencing of CrWRKY42 in citrus fruits indicated that CrWRKY42 positively regulated chlorophyll degradation and carotenoid biosynthesis by synergistically activating the expressions of genes involved in both pathways. In conclusion, our data revealed that CrWRKY42 acted as a positive regulator of chlorophyll degradation and carotenoid biosynthesis to alter the conversion of citrus fruit color. Our findings provide insight into the complex transcriptional regulation of chlorophyll and carotenoid metabolism during fruit ripening. One Sentence SummaryThe CrWRKY42 transcription factor coordinates chlorophyll degradation and carotenoid biosynthesis by directly regulating genes involved in these pathways to alter the conversion of citrus fruit color.

BackgroundTransposable elements (TEs) are major components of plant genomes. Despite being regarded as "junk DNA" at first, TEs play important roles for the organisms they are found in. The most obvious and easily recognizable effects caused by TEs result from their mobility, which can disrupt coding sequences or promoter regions. However, with the recent advances in transcriptomics, it is becoming increasingly evident that TEs can act as an additional layer of gene expression regulation through a number of processes, which can involve production of non-coding RNAs. Here, we describe how Tnt1, a stress-responsive LTR-retrotransposon, interferes with gene expression and modulate a number of developmental aspects in tobacco. ResultsThrough an RNAi approach, we generated tobacco (HP) lines knocked-down for Tnt1 expression. Quantitative RT-PCR experiments confirm that Tnt1 is downregulated in HP lines after ethylene exposure. A RNA-seq experiment was performed and through two independent bioinformatic approaches (with different stringencies) we found 932 and 97 differentially expressed genes in HP lines. A number of phenotypes were observed in such lines, namely lesion mimicry in leaves, underdevelopment of the root system, overproduction of root hairs and early loss of seed viability. Folding prediction of part of the Tnt1 mRNA reveals putative stem-loop secondary structures containing transcriptional regulation sequences, suggesting it could be a source of small RNAs. We also propose a model to explain the Tnt1 expression in both homeostatic and stress conditions, and how it could interact with stress-responsive genes. ConclusionsOur results are consistent that interferences with Tnt1 transcript levels correlate with transcriptomic and phenotypic changes, suggesting a functional role for this element during plant development and stress response.

Iron (Fe) is an essential trace element for plants. When suffering from Fe deficiency, plants modulate the expression of Fe deficiency responsive genes. POPEYE (PYE) is a key bHLH transcription factor involved in Fe homeostasis. However, the molecular mechanism of PYE regulating the Fe deficiency response remains elusive. We found that the over-expression of PYE attenuates the expression of Fe deficiency responsive genes. PYE directly represses the transcription of bHLH Ib genes (bHLH38, bHLH39, bHLH100, and bHLH101) by associating with their promoters. Although PYE contains an Ethylene response factor-associated Amphiphilic Repression (EAR) motif, it does not interact with the transcriptional corepressors TOPLESS/TOPLESS-RELATED (TPL/TPRs). Subcellular localization analysis indicated that PYE localizes in both the cytoplasm and nucleus. PYE contains a Nuclear Export Signal (NES) which is required for the cytoplasmic localization of PYE. The mutation of NES amplifies the repression function of PYE, resulting in downregulation of Fe deficiency responsive genes. Co-expression assays indicated that bHLH IVc members (bHLH104, bHLH105/ILR3, and bHLH115) facilitate the nuclear accumulation of PYE. Conversely, PYE indirectly represses transcription activation ability of bHLH IVc. Additionally, PYE directly negatively regulates its own transcription. This study provides insights into the complicated Fe deficiency response signaling pathway and enhances the understanding of PYE functions. Short summaryPYE is a negative regulator of Fe homeostasis; however, it was still unclear how PYE integrates the Fe deficiency response signaling. Our study shows that conditional nuclear localization of PYE is crucial for Fe homeostasis. PYE not only negatively regulates FIT-dependent Fe uptake genes by directly targeting bHLH Ib genes, but also negatively regulates its own expression.

Recently, the siRNAs pathways, and especially reproductive phasiRNAs, have attracted attention in eudicots since their biological roles are still unknown and their biogenesis took different evolutionary pathways compared to monocots. In this work, we used Coffea arabica L., a recently allotetraploid formed from the hybridization of C. canephora and C. eugenioides unreduced gametes, to explore microsporogenesis and small RNAs related pathways in a eudicot crop. First, we identified the microsporogenesis stages during anther development revealing that pre-meiosis occurs in anthers of 1.5 mm inside floral buds (FBs), whereas meiosis between 1.5 and 4.2 mm FBs, and post-meiosis in FBs larger than 4.2mm. These stages coincide with the Brazilian winter, a period of FBs reduced growth which suggests temperature sensitivity. Next, we identified and quantified the expression of reproductive 21- and 24-nt phasiRNAs during coffee anther development together with their canonical and novel miRNA triggers, and characterized the DCL and AGO families. Our results showed that the pattern of reproductive phasiRNA abundance in C. arabica is unique among described eudicots and the canonical trigger car-miR2275 is involved in the processing of both 21 and 24 nt phasiRNAs. Fourteen DCL genes were identified, but DCL5, related to phasiRNA biosynthesis in monocots, was not according to its specificity for monocots. Thus, our work explored the knowledge gap about microsporogenesis and related siRNAs pathways in coffee, contributing to the control of reproductive development and to the improvement of fertility in eudicots.