Plant Cell Reports

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Online ISSN: 1432-203X
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Drought survival strategies like drought resistance and drought escape involve several morphological and biochemical adaptations in plants. Drought resistance can be further categorized into drought tolerance and drought avoidance mechanisms. Drought tolerance involves responses like delayed senescence, induction of drought-responsive proteins (DREB (dehydration-responsive element binding), LEA (late-embryogenesis abundant), MYB, NAC (NAM, ATAF, and CUC), NCED (nine-cis-epoxycarotenoid dioxygenase 3)) and hormonal pathways (ABA, BR, Auxin), antioxidant and osmoprotectant accumulation, and regulation of cuticular thickness. Drought avoidance responses involve morphological adaptations like deeper roots, late flowering, stomatal closure, and inhibition of stomatal development, conductance, transpiration together with a reduction in leaf area. Drought escape is a separate strategy used by plants to complete their life cycle before the onset of drought that involves early flowering and grain filling
The interplay between light and drought signaling regulates stomatal development and dynamics, root development, and senescence to acquire drought resistance. A Drought stress promotes synthesis and accumulation of ABA and BR (brassinosteroids). BR and COP1 (constitutive photomorphogenic 1) oppositely regulate YDA (YODA) that further inhibits the bHLH (basic helix–loop–helix) transcription factors like SPCH (SPEECHLESS), MUTE, and FAMA, which control stomatal development. SPCH and MUTE are also inhibited by ABA. AN3 (Angustifolia 3), an integrator of light and drought signaling, is promoted by light and repressed by drought. After being induced, AN3 suppresses both YDA and COP1 to promote guard cell development. YDA might act as a convergence point between light and drought signaling. B cry1/cry2 and phototropins activate stomatal opening genes by suppressing the COP1-mediated inhibition of MYB60 that promotes stomatal opening. During drought, ABA inhibits MYB60 to prevent the opening of stomata. H⁺-ATPase also gets regulated oppositely by ABA and blue light. ABA inhibits light mediated activation of H+-ATPase to promote stomatal closure under drought condition. C Red light promotes primary root elongation through phyB that promotes PER1 (peroxidase 1) production via ABI5 by promoting the biosynthesis of ABA. PER1 inhibits ROS production in the root that ultimately leads to increased primary root growth. ABI5 also regulates primary root growth negatively by inhibiting PIN1. ABA- as well as light-activated HY5 can stimulate MIZ1 (MIZU-KUSSEI1) to promote root hydrotropic bending. D Survival under drought involves the regulation of senescence in plants. In regulating senescence, BBX22 and PIF4 act as a convergence point as their signaling pathways are modulated by both drought-induced ABA and red light. BBX22 on getting activated by ABA and red light induces genes coding for chlorophyll-degrading enzymes SGR/NYE/NYC1/NYC2 via ABF4 (ABA response element-binding factor 4) to promote chlorophyll degradation in leaves during senescence. PIF4 and PIF5 are inhibited by phyB, and together with ABI5, and EEL activate senescence master regulator gene ORE1, which further activates chlorophyll degradation genes like SGR1 (shoot gravitropism 1), NYC1 (non-yellow coloring 1). Black arrows indicate promotion, and T represents inhibition. Broken arrows in C indicate the involvement of other factors in between the two signaling components
Drought is one of the most critical stresses, which causes an enormous reduction in crop yield. Plants develop various strategies like drought escape, drought avoidance, and drought tolerance to cope with the reduced availability of water during drought. Plants adopt several morphological and biochemical modifications to fine-tune their water-use efficiency to alleviate drought stress. ABA accumulation and signaling plays a crucial role in the response of plants towards drought. Here, we discuss how drought-induced ABA regulates the modifications in stomatal dynamics, root system architecture, and the timing of senescence to counter drought stress. These physiological responses are also regulated by light, indicating the possibility of convergence of light- and drought-induced ABA signaling pathways. In this review, we provide an overview of investigations reporting light-ABA signaling cross talk in Arabidopsis as well as other crop species. We have also tried to describe the potential role of different light components and their respective photoreceptors and downstream factors like HY5, PIFs, BBXs, and COP1 in modulating drought stress responses. Finally, we highlight the possibilities of enhancing the plant drought resilience by fine-tuning light environment or its signaling components in the future.
Keymessage CbSE overexpression increased stigmasterol levels and altered plant morphology. The genes upstream and downstream of CbSE were found to be upregulated, which confirms its regulatory role in the saponin biosynthetic pathway. Abstract Chlorophytum borivilianum is a high-value medicinal plant with many promising preclinical applications that include saponins as a major active ingredient. Squalene epoxidase (SE) is one of the major rate-limiting enzymes of the saponin biosynthetic pathway. Here, we functionally characterized C. borivilianum SE (CbSE) by over-expressing heterologously in Nicotiana tabacum. The heterologous expression of CbSE resulted in stunted pant growth with altered leaf and flower morphology. Next, RT-qPCR analysis of transgenic plants overexpressing CbSE revealed increased expression levels of Cycloartenol synthase (CAS), Beta amyrin synthase (βAS), and cytochrome P450 monooxygenase 51 (CYP51) (Cytochrome P450), which encode key enzymes for triterpenoid and phytosterol biosynthesis in C. borivilianum. Further, Methyl Jasmonate (MeJa) treatment upregulated Squalene synthase (SQS), SE, and Oxidosqualene cyclases (OSCs) to a significant level. GC–MS analysis of the leaf and hairy roots of the transformants showed an increased stigmasterol content (0.5–1.0 fold) compared to wild type (WT) plants. These results indicate that CbSE is a rate-limiting gene, which encodes an efficient enzyme responsible for phytosterol and triterpenoid production in C. borivilianum.
Key message An engineered selectable marker combining herbicide resistance and yellow fluorescence contributes to the characterization of male-sterile phenotype in wheat, the severity of which correlates with expression levels of a synthetic Ms2 gene. Abstract Genetic transformation of wheat is conducted using selectable markers, such as herbicide and antibiotic resistance genes. Despite their proven effectiveness, they do not provide visual control of the transformation process and transgene status in progeny, which creates uncertainty and prolongs screening procedures. To overcome this limitation, this study developed a fusion protein by combining gene sequences encoding phosphinothricin acetyltransferase and mCitrine fluorescent protein. The fusion gene, introduced into wheat cells by particle bombardment, enabled herbicide selection, and visual identification of primary transformants along with their progeny. This marker was then used to select transgenic plants containing a synthetic Ms2 gene. Ms2 is a dominant gene whose activation in wheat anthers leads to male sterility, but the relationship between the expression levels and the male-sterile phenotype is unknown. The Ms2 gene was driven either by a truncated Ms2 promoter containing a TRIM element or a rice promoter OsLTP6. The expression of these synthetic genes resulted in complete male sterility or partial fertility, respectively. The low-fertility phenotype was characterized by smaller anthers than the wild type, many defective pollen grains, and low seed sets. The reduction in the size of anthers was observed at earlier and later stages of their development. Consistently, Ms2 transcripts were detected in these organs, but their levels were significantly lower than those in completely sterile Ms2TRIM::Ms2 plants. These results suggested that the severity of the male-sterile phenotype was modulated by Ms2 expression levels and that higher levels may be key to activating total male sterility.
CRISPR/Cas9-mediated knockout of the polyester synthase-like genes At1G73750 and MSAD_264347 delays flowering time in Arabidopsis thaliana and Medicago sativa (alfalfa). Homozygous wild-type (Col-0) and mutant (SALK_002005.55.10.X) lines of At1G73750 were obtained from the Arabidopsis Biological Resource Center ( a In comparison with the parental wild-type line (Col-0), the mutant line (At1G73750) displays a clear flowering delay under optimal growth conditions (Soto et al. 2010). b Specifically, mutant plants flower 6 days after wild-type plants. c The leaf number, d lipid peroxidation e and anthocyanin (Fig. 1e) of wild-type and mutant lines (Ayub et al. 2009; Soto et al. 2011) were examined in 56-day-old plants. All values are means + SEM (n = 9–30). Significant differences between wild-type and mutant plants were analyzed with ANOVA followed by Dunnett's multiple comparisons test (*p < 0.05, ****p < 0.0001). g Genotype of wild-type and alfalfa-edited (MSAD_264347) lines. In this picture, we show the nucleotide sequence of four wild-type alleles of the MSAD_264347 gene from the regenerative wild-type alfalfa line C23 (alfalfa is a tetraploid species), the first exon of the MSAD_264347 gene, the first guide (sgRNA1), the sequence recognized by the Cas9 nuclease (PAM sequences), the short deletions (> 6 nucleotides) within these alleles in the CRISPR/Cas9-editing event line and the frameshift mutation induced by these deletions. h In comparison with the parental wild-type line, the homozygous mutant line displayed an obvious flowering delay under soil conditions. i A pool of nine heterozygous mutant lines containing different doses of edited alleles (AAAa, AAaa and Aaaa) and the homozygous mutant line (aaaa) flowered 6 and 7 days after wild-type plants (AAAA), respectively. Wild-type and truncated MSAD_264347 alleles are represented by “A” and “a”, respectively. All values are means + SEM (n = 9–26). Significant differences between non-edited and edited plants were analyzed with ANOVA followed by Dunnett's multiple comparisons test (*p < 0.5, **p < 0.01)
Key message T-DNA and CRISPR/Cas9-mediated knockout of polyester synthase-like genes delays flowering time in Arabidopsis thaliana and Medicago sativa (alfalfa). Thus, we here present the first report of edited alfalfa with delayed flowering.
Key message Chrysanthemum morifolium MYB3 factors are transcriptional activators for the regulation of flavonol biosynthesis. Abstract Flavonol was not only the critical secondary metabolite participating in the growth and development of plants but also the main active ingredient in medicinal chrysanthemum. However, few pieces of research revealed the transcriptional regulation of flavonol biosynthesis in Chrysanthemum morifolium. Here, we isolated two CmMYB3 transcription factors (CmMYB3a and CmMYB3b) from the capitulum of Chrysanthemum morifolium cv ‘Hangju’. According to the sequence characteristics, the CmMYB3a and CmMYB3b belonged to the R2R3-MYB subgroup 7, whose members were often reported to regulate flavonol biosynthesis positively. CmMYB3a and CmMYB3b factors were identified to localize in the nucleus by subcellular localization assay. Besides, both of them have obvious transcriptional self-activation activity in their C-terminal. After the overexpression of CmMYB3 genes in Nicotiana benthamiana and Arabidopsis thaliana, the flavonol contents in plants were increased, and the expression of AtCHS, AtCHI, AtF3H, and AtFLS genes in A. thaliana was also improved. Interestingly, the CmMYB3a factor had stronger functions in improving flavonol contents and related gene expression levels than CmMYB3b. The interaction analysis between transcription factors and promoters suggested that CmMYB3 could bind and activate the promoters of CmCHI and CmFLS genes in C. morifolium, and CmMYB3a also functioned more powerfully. Overall, these results indicated that CmMYB3a and CmMYB3b work as transcriptional activators in controlling flavonol biosynthesis.
Gene expression analysis and subcellular localization of LiMYB108. The expressions of LoTPS1(A) and LiMYB108 (B) at different light intensity levels (0, 200, and 600 μmol m⁻² s⁻¹) were detected by qRT-PCR. C Expression profiles of LiMYB108 during the four developmental stages. S1 (budding stage), S2 (initial flowering stage), S3 (full-blooming stage), and S4 (decay stage). D Expression levels of LiMYB108 in night tissues. The error bars represent the means of three biological replicates ± SDs, and the asterisks indicate significant differences according to the t test (*P < 0.05; **P < 0.01). E Subcellular localization of LiMYB108. The plasmid pNC-Cam1304-LiMYB108-GFP was co-infiltrated with the nuclear marker NF-YA4-mCherry. Green and red fluorescence were observed by confocal microscopy 3 days after infection
Effect of silencing LiMYB108 on the three main monoterpenoids and gene expression levels. A Total ion current (TIC) chromatograms of the flower fragrance compounds emitted from Lilium ‘Siberia’. The peaks correspond to myrcene, ocimene, and linalool. The first column shows the TIC of ‘Siberia’ infected with pNC-TRV2 (control) and the second column shows the TIC of ‘Siberia’ infected with pNC-TRV2-LiMYB108.B qRT-PCR results of the LiMYB108 mRNA levels in TRV2-LiMYB108 petals compared with control pTRV2 petals. C qRT-PCR results of LoTPS1 mRNA levels in the petals of LiMYB108-silenced plants compared with control plants. D Release of the three main monoterpenoids in the petals of LiMYB108-silenced plants compared with control plants. *P < 0.05, **P < 0.01. Data are presented as mean ± SD, n = 3
Overexpression of LiMYB108 in Lilium ‘Siberia’. A Total ion current (TIC) chromatograms of the flower fragrance compounds emitted from Lilium ‘Siberia’. The peaks correspond to myrcene, ocimene, and linalool. The first column shows the TIC of ‘Siberia’ infected with pNC-Cam3304-35S (control), and the second column shows the TIC of ‘Siberia’ infected with pNC-Cam3304-35S-LiMYB108.B qRT-PCR results of the LiMYB108 mRNA levels in pNC-Cam3304-35S-LiMYB108 petals compared with control petals. C qRT-PCR results of LoTPS1 mRNA levels in the petals of LiMYB108-overexpressed plants compared with control plants. D Release of the three main monoterpenoids in the petals of LiMYB108 overexpressed plants compared with control plants. *P < 0.05, **P < 0.01. Data are presented as mean ± SD, n = 3
Interaction between LiMYB108 and the LoTPS1 promoter. A Schematic diagrams of MYB binding motifs in LoTPS1 promoter. B LiMYB108 binding to the LoTPS1 promoter in the yeast one-hybrid system. The empty prey vector (AD) was used as the negative control. C Schematic representation of the constructed dual LUC reporter system. D Live imaging (left) and quantitative analysis (right) of transcriptional repression of the LoTPS1 pro 1 by LiMYB108. E Live imaging (left) and quantitative analysis (right) of transcriptional repression of the LoTPS1 pro 2 by LiMYB108. The LUC: LoTPS1 pro construct was co-infiltrated with SK:LiMYB108 or the SK empty vector in N. benthamiana leaves. The experiments were replicated three times. A representative image of an N. benthamiana leaf 3 days after infiltration is shown. *P < 0.05, **P < 0.01. The mean ± SD correspond to three replicates (n = 3). F EMSA of LiMYB108 binding to the first cite of LoTPS1 promoter. G EMSA of LiMYB108 binding to the second cite of LoTPS1 promoter
Key message We find that the MYB family transcription factor, LiMYB108, has a novel function to regulate the floral fragrance affected by light intensity. Abstract Floral fragrance determines the commercial value of flowers and is influenced by many environmental factors, especially light intensity. However, the mechanism by which light intensity affects the release of floral fragrance is unclear. Here, we isolated an R2R3-type MYB transcription factor LiMYB108, the expression of which was induced by light intensity and located in the nucleus. Light of 200 and 600 μmol m⁻¹ s⁻¹ significantly increased the expression of LiMYB108, which was consistent with the improving trend of monoterpene synthesis under light. Virus-induced gene silencing (VIGS) of LiMYB108 in Lilium not only significantly inhibited the synthesis of ocimene and linalool, but also decreased the expression of LoTPS1; however, transient overexpression of LiMYB108 exerted opposite effects. Furthermore, yeast one-hybrid assays, dual-luciferase assays, and electrophoretic mobility shift assays (EMSA) demonstrated that LiMYB108 directly activated the expression of LoTPS1 by binding to the MYB binding site (MBS) (CAGTTG). Our findings demonstrate that light intensity triggered the high expression of LiMYB108, and then LiMYB108 as a transcription factor to activate the expression of LoTPS1, thus promoting the synthesis of the ocimene and linalool, which are important components of floral fragrance. These results provide new insights into the effects of light intensity on floral fragrance synthesis.
Key message PvLBD12 enhanced the salt tolerance by increasing proline accumulation, improving K⁺ accumulation, and decreasing reactive oxygen species level in switchgrass. Abstract Abiotic stresses are the serious factors which limit plant development and productivity and restrict the agricultural economy. It is important, therefore, to understand the mechanism of abiotic tolerance in plants. Lateral organ boundaries domain (LBD) proteins as plant-specific transcription factors play important function in plant lateral organ development, plant regeneration, and abiotic stress. In our study, we identify 69 LBD members from switchgrass genome-wide sequences and classify them based on their homology with LBD proteins in Arabidopsis. RT-qPCR showed that PvLBD genes had different expression patterns under abiotic stress conditions, indicating that they play important roles in various stress. PvLBD12 was selected as a candidate gene for further functional analysis because it had the highest expression level under salt stress. Overexpression of PvLBD12 enhanced salt tolerance by altering a wide range of physiological responses (like increased proline accumulation, reduced malondialdehyde production, improved K⁺ accumulation, and reduced Na⁺ absorption) in switchgrass. Some stress response genes such as proline biosynthesis gene PvP5CS1, vacuolar Na⁺(K⁺)/H⁺ antiporter gene PvNHX1, two key ROS-scavenging enzyme genes PvCAT and PvSOD were all upregulated in PvLBD12 overexpression lines. Taken together, PvLBD12 plays a pivotal role in response to salt stress by increasing proline accumulation, improving K⁺ accumulation, reducing Na⁺ absorption, and decreasing reactive oxygen species level. It will be better to understand the potential biological functions of LBD genes in other plants.
SINA (Seven in absentia) proteins in the subtype of E3 ubiquitin ligase family play a crucial role in plant growth and development. However, their functions in response to salt and osmotic stresses in oil crops are still largely unknown. In this study, a total number of 23 BnaSINAs were identified in the rapeseed genome. Chromosome location and collinear relationship analyses revealed that they were unevenly distributed on 13 chromosomes, and have gone through 22 segmental duplication events under purifying selection. Phylogenetic and gene structural analyses indicated that they belonged to five main groups, and those in the same subgroup showed similar gene structure. All BnaSINAs were predicted to form homo- or heterodimers. Except BnaSINA7, BnaSINA11, BnaSINA17 and BnaSINA18, which lacked the N-terminal RING finger, all BnaSINAs contained a conserved C-terminal SINA domain, a typical structural feature of the RING-type E3 ligase family. Transcriptional expression analyses demonstrated that most BnaSINAs were ubiquitously expressed in roots, stems, leaves, flowers, pods and seeds, and all were responsive to salt and osmotic stresses. Further, yeast two-hybrid and Arabidopsis mutant complementation analyses demonstrated that BnaSINA4 interacted with BnaSINA17 to form heterodimer, and expression of BnaSINA17 in the Arabidopsis sina2 mutant restored its growth resistance to salt and osmotic stresses. Our findings provide an important genetic foundation for the functional elucidation of BnaSINAs and a novel gene resource for the breeding of new oil crop cultivars with improved abiotic stress resistance.
Overview of the implications of seed priming technologies throughout basic, translational, and applied research, highlighting the most relevant deliverables of each phase and stating the main open questions driving future developments
The rehydration–dehydration cycle as a schematic representation of the treatment (controlled seed imbibition followed by desiccation or dry-back) routinely applied in standard seed vigorization protocols. Primed seeds undergo post-priming germination. The different steps of the rehydration–dehydration cycle can be regarded as potential sources of novel seed quality hallmarks, specified as genes, proteins, metabolites
Molecular mechanisms of seed priming. The main drawbacks of the technique, overpriming and reduced shelf-life of primed seeds are also represented
Representation of the cellular and subcellular sources of the different seed quality hallmarks currently under investigation. ROS reactive oxygen species, n nucleus, nu nucleolus, mt mitochondria, lm lipid membrane, r ribosome
Key message The potential of seed priming is still not fully exploited. Our limited knowledge of the molecular dynamics of seed pre-germinative metabolism is the main hindrance to more effective new-generation techniques. Abstract Climate change and other recent global crises are disrupting food security. To cope with the current demand for increased food, feed, and biofuel production, while preserving sustainability, continuous technological innovation should be provided to the agri-food sector. Seed priming, a pre-sowing technique used to increase seed vigor, has become a valuable tool due to its potential to enhance germination and stress resilience under changing environments. Successful priming protocols result from the ability to properly act on the seed pre-germinative metabolism and stimulate events that are crucial for seed quality. However, the technique still requires constant optimization, and researchers are committed to addressing some key open questions to overcome such drawbacks. In this review, an update of the current scientific and technical knowledge related to seed priming is provided. The rehydration–dehydration cycle associated with priming treatments can be described in terms of metabolic pathways that are triggered, modulated, or turned off, depending on the seed physiological stage. Understanding the ways seed priming affects, either positively or negatively, such metabolic pathways and impacts gene expression and protein/metabolite accumulation/depletion represents an essential step toward the identification of novel seed quality hallmarks. The need to expand the basic knowledge on the molecular mechanisms ruling the seed response to priming is underlined along with the strong potential of applied research on primed seeds as a source of seed quality hallmarks. This route will hasten the implementation of seed priming techniques needed to support sustainable agriculture systems.
Key message We reported the mitochondrial genome of Taraxacum mongolicum for the first time. Five pairs of repeats that can mediate recombination were validated, leading to multiple conformations of genome. Abstract Taraxacum mongolicum belongs to the Asteraceae family and has important pharmaceutical value. To explore the possible interaction between the organelle genomes, we assembled the complete mitochondrial genome (mitogenome) of T. mongolicum using Illumina and Oxford Nanopore sequencing data. This genome corresponded to a circular molecule 304,467 bp long. It encodes 52 unique genes including 31 protein-coding, 3 ribosomal RNA (rRNA) and 18 transfer RNA (tRNA) genes. In addition to the single circular conformation, the existence of alternative conformations mediated by five repetitive sequences in the mitogenome was identified and validated. Recombination mediated by the inverted repeats resulted in two conformations. Conversely, recombination mediated by the two direct repeats broke one large circular molecule into two subgenomic circular molecules. Furthermore, we identified 12 homologous fragments by comparing the sequences of mitogenome and plastome, including eight complete tRNA genes. Lastly, we identified a total of 278 RNA-editing sites in protein-coding sequences based on RNA-seq data. Among them, cox1 and nad5 gene has the most sites (21), followed by the nad2 gene with 19 sites. We successfully validated 213 predicted RNA-editing sites using PCR amplification and Sanger sequencing. This project reported the first mitogenome of T. mongolicum and demonstrated its multiple conformations generated by repeat-mediated recombination. This genome could provide critical information for the molecular breeding of T. mongolicum, and also be used as a reference genome for other species of the genus Taraxacum.
Key message Sequestering microRNA396 by overexpression of MIM396 enhanced alfalfa resistance to Spodoptera litura larvae, which may be due to increased lignin content and enhanced low-molecular weight flavonoids and glucosinolates biosynthesis. Abstract Alfalfa (Medicago sativa), the most important leguminous forage crop, suffers from the outbreak of defoliator insects, especially Spodoptera litura, resulting in heavy losses in yield and forage quality. Here, we found that the expression of alfalfa microRNA396 (miR396) precursor genes and mature miR396 was significantly up-regulated in wounding treatment that simulates feeding injury by defoliator insects. To verify the function of miR396 in alfalfa resistance to insect, we generated MIM396 transgenic alfalfa plants with significantly down-regulated miR396 expression by Agrobacterium-mediated genetic transformation. The MIM396 transgenic alfalfa plants exhibited improved resistance to Spodoptera litura larvae with increased lignin content but decreased JA accumulation. Most of the miR396 putative target GRF genes were up-regulated in MIM396 transgenic lines, and responded to the wounding treatment. By RNA sequencing analysis, we found that the differentially expressed genes related to insect resistance between WT and MIM396 transgenic plants mainly clustered in biosynthesis pathways in lignin, flavonoids and glucosinolates. In addition to the phenotype of enhanced insect resistance, MIM396 transgenic plants also displayed reduced biomass yield and forage quality. Our results broaden the function of miR396 in alfalfa and provide genetic resources for studying alfalfa insect resistance.
Significant ncRNAs implicated in callus induction and plant cell dedifferentiation in different plant species. Noteworthy studies have ascertained that multiple classes of ncRNAs play a relevant role in the transition of somatic cells into totipotent cells during callus formation. Among them, some of the most relevant species analyzed on this topic are Zea mays L. (maize), Solanum lycopersicum L. (tomato), Oryza sativa L. (rice), Gossypium hirsutum L. (cotton), Camellia sinensis (L.) Kuntze (tea plant), and Arabidopsis thaliana. As depicted, most of the ncRNAs associated with callus induction belong to the miRNA class; notwithstanding, siRNAs and lncRNAs have been related to this biological process in Z. mays and O. sativa, respectively
Potential applications of ncRNA-mediated technologies in callus culture. The use of emerging biotechnological approaches (e.g., genome editing and artificial plant RNAs) considering both ncRNAs and genes involved in callus induction, as well as the analysis of the ncRNA expression profile of other significant plants during callus induction and plant cell dedifferentiation could potentially enhance the production of calli of different species. Consequently, the proper understanding and application of such approaches might be useful to boost the manufacture of bioactive compounds, biofuels, plant-based vaccines, and antibodies and increase the production of both agricultural and ornamental plants
Biological functions of ncRNAs belonging to different plant species during callus induction and cell dedifferentiation
Key message Plant regulatory noncoding RNAs (ncRNAs) have emerged as key modulators of gene expression during callus induction. Their further study may promote the design of innovative plant tissue culture protocols. Abstract The use of plants by humans has recently taken on a new and expanding insight due to the advent of genetic engineering technologies. In this context, callus cultures have shown remarkable potential for synthesizing valuable biomolecules, crop improvement, plant micropropagation, and biodiversity preservation. A crucial stage in callus production is the conversion of somatic cells into totipotent cells; compelling evidence indicates that stress factors, transcriptional regulators, and plant hormones can trigger this biological event. Besides, posttranscriptional regulators of gene expression might be essential participants in callus induction. However, research related to the analysis of noncoding RNAs (ncRNAs) that modulate callogenesis and plant cell dedifferentiation in vitro is still at an early stage. During the last decade, some relevant studies have enlightened the fact that different classes of ncRNAs, such as microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs) are implicated in plant cell dedifferentiation through regulating the expression levels of diverse gene targets. Hence, understanding the molecular relevance of these ncRNAs in the aforesaid biological processes might represent a promising source of new biotechnological approaches for callus culture and plant improvement. In this current work, we review the experimental evidence regarding the prospective roles of ncRNAs in callus induction and plant cell dedifferentiation to promote this field of study.
Key message D129 is an EMS-induced mutant with dwarf phenotype, which has important breeding potential to cultivate new varieties suitable for high-density planting in maize Abstract Plant height is one of the important agronomic traits that affecting maize planting density, identification of superior dwarf mutants can provide important genetic materials for breeding new varieties suitable for high-density planting. In this study, we identified a dwarf mutant, d129, from maize EMS-induced mutant population. Gene mapping indicated that a G-to-A transition in the second exon of the br2 gene was responsible for the dwarf phenotype of the d129 mutant using MutMap method, which was further validated through allelism testing. Compared with WT plants, the average plant height and ear height of d129 were reduced by 26.67% and 39.43%, respectively, mainly due to a decrease in internode length. Furthermore, the d129 mutant exhibited increased internode diameter, which is important for increasing planting density due to the lodging resistance may be enhanced. Endogenous hormone measurement demonstrated that the contents of IAA and GA3 in the internode of the mutant were significantly lower than that of WT plants. RNA-seq analysis indicated that at least fifteen auxin-responsive and signaling-related genes exhibited differential expression, and some genes involved in cell development and other types of hormone signaling pathways, were also identified from the differential expressed genes. These genes may be related to the reduced hormone contents and decreased elongation of internode cells of the d129 mutant. Our study provided a novel dwarf mutant which can be applied in maize breeding to cultivate new varieties suitable for high-density planting.
Key message We identified a miraculin-like protein (NaMLP) who is a new Kunitz trypsin inhibitor regulated synergistically by JA and ethylene signals and confers Spodoptera litura resistance in wild tobacco Nicotiana attenuata. The findings revealed a new source of trypsin inhibitor activities after herbivory, and provide new insights into the complexity of the regulation of trypsin inhibitor-based defense after insect herbivore attack. Abstract Upon insect herbivore attack, wild tobacco Nicotiana attenuata accumulates trypsin protease inhibitor (TPI) activities as a defense response from different protease inhibitor (PI) coding genes, including WRKY3-regulated NaKTI2, and JA-dependent NaPI. However, whether any other TPI gene exists in N. attenuata is still unclear. A miraculin-like protein gene (NaMLP) was highly up-regulated in N. attenuata after Alternaria alternata infection. However, silencing or overexpression of NaMLP had no effect on the lesion diameter developed on N. attenuata leaves after A. alternata inoculation. Meanwhile, the transcripts of NaMLP could be induced by wounding and amplified by Spodoptera litura oral secretions (OS). S. litura larvae gained significantly more biomass on NaMLP-silenced plants but less on NaMLP overexpressed plants. Although NaMLP showed low sequence similarity to NaKTI2, it had conserved reaction sites of Kunitz trypsin inhibitors, and exhibited TPI activities when its coding gene was overexpressed transiently or stably in N. attenuata. This was consistent with the worst performance of S. litura larvae on NaMLP overexpressed lines. Furthermore, NaMLP-silenced plants had reduced TPI activities and better S. litura performance. Finally, OS-elicited NaMLP was dramatically reduced in JA-deficient AOC silencing and ethylene-reduced ACO-silencing plants, and the expression of NaMLP could be significantly induced by methyl jasmonate or ethephon alone, but dramatically amplified by co-treatment of both methyl jasmonate and ethephon. Thus, our results demonstrate that in addition to JA-regulated NaPI, and WRKY3/6-dependent NaKTI2, N. attenuata plants also up-regulates TPI activities via NaMLP, which confers S. litura resistance through JA and ethylene signaling pathways in a synergistic way.
The seed setting rate and 1000-grain weight of Huaidao5 and Huaidao9. Values are presented as means ± SD (N = 10). Data differences among groups are evaluated by Duncan's multiple range tests. Different letters indicate significant differences among groups (p < 0.05). H5: Huaidao5, H9: Hudaidao9, CT: cold treatment, CK: control
The number of differentially expressed genes in treatment (CT/CK), cultivar (H5/H9) and tissue (panicles/leaves)
Hierarchical cluster tree revealing co-expression modules identified by WGCNA. Each leaf in the tree represents a gene. The main tree branches constitute 36 modules, labeled with different colors
Module–trait association. Correlation between each module and traits (treatment, cultivar, tissue and sample) revealed with the correlation coefficient and p value; red means positive correlation and blue indicates negative correlation. Samples were named in the order of cultivar, tissue and treatment, such as the panicle sample of Huaidao5 under cold treatment, which was referred to as H5PCT. H5: Huaidao5, H9: Hudaidao9, P: panicles, L: leaves, CT: cold treatment, CK: control
The core networks obtained from the interaction network analysis with known-core genes for treatment-specific (a: dark gray, b: yellow) module. The size of the nodes indicate the degree of connectivity in the network. Nodes in red color are known-core genes. The square nodes represent candidates related to cold response
Key message The resistance of Huaidao5 results from the high constitutive expression of tolerance genes, while that of Huaidao9 is due to the cold-induced resistance in flag leaves and panicles. Abstract The regulation mechanism of rice seedlings’ cold tolerance is relatively clear, and knowledge of its underlying mechanisms at the reproductive stage is limited. We performed differential expression and co-expression network analyses to transcriptomes from panicle and flag leaf tissues of a cold-tolerant cultivar (Huaidao5), and a sensitive cultivar (Huaidao9), under reproductive-stage cold stress. The results revealed that the expression levels of genes in stress-related pathways such as MAPK signaling pathway, diterpenoid biosynthesis, glutathione metabolism, plant–pathogen interaction and plant hormone signal transduction were constitutively highly expressed in Huaidao5, especially in panicles. Moreover, the Hudaidao5’s panicle sample-specific (under cold) module contained some genes related to rice yield, such as GW5L, GGC2, SG1 and CTPS1. However, the resistance of Huaidao9 was derived from the induced resistance to cold in flag leaves and panicles. In the flag leaves, the responses included a series of stress response and signal transduction, while in the panicles nitrogen metabolism was severely affected, especially 66 endosperm-specific genes. Through integrating differential expression with co-expression networks, we predicted 161 candidate genes (79 cold-responsive genes common to both cultivars and 82 cold-tolerance genes associated with differences in cold tolerance between cultivars) potentially affecting cold response/tolerance, among which 85 (52.80%) were known to be cold-related genes. Moreover, 52 (65.82%) cold-responsive genes (e.g., TIFY11C, LSK1 and LPA) could be confirmed by previous transcriptome studies and 72 (87.80%) cold-tolerance genes (e.g., APX5, OsFbox17 and OsSTA109) were located within QTLs associated with cold tolerance. This study provides an efficient strategy for further discovery of mechanisms of cold tolerance in rice.
Enhanced chloroplast–chloroplast physical interactions in cTP-mCherry-expressing cells. a Confocal microscope images of N. benthamiana leaves transiently expressing constructs encoding cTP (the N-terminal 50 aa of OEP7) fused with sfGFP, mVenus, or mCherry (cTP-sfGFP, cTP-mVenus, or cTP-mCherry, respectively). CHL, chlorophyll autofluorescence. Epidermal cells were observed 1.5 days after infiltration. Bars, 10 µm. b Mitochondria and peroxisome localization in the presence or absence of cTP-mCherry. Constructs encoding the mitochondria marker (mt-GFP) or the peroxisome marker (po-mCerulean) were transiently expressed with or without cTP-mCherry. Fluorescent signals are also seen in the cytosol due to mistargeting of mt-GFP and po-mCerulean. Epidermal cells of N. benthamiana leaves were observed 1.5 days after infiltration. Bars, 10 µm. c Immunoblot analysis of fluorescent proteins fused with cTP. Total proteins were extracted from N. benthamiana leaves transiently expressing cTP-sfGFP, cTP-mVenus, or cTP-mCherry 1.5 days after infiltration with Laemmli sample buffer with or without the reducing agent dithiothreitol (DTT). Samples extracted with the sample buffer without DTT were analyzed without boiling. Coomassie brilliant blue (CBB) staining shows blots of Rubisco large subunits (RbcL) as a loading control. Red dots, non-denatured form; red asterisks, denatured form; red line, multimers
Generation of chloroplast-glued Arabidopsis plants. a Confocal microscope images of Arabidopsis leaves expressing cTP-sfGFP or cTP-mCherry under the control of the CaMV35S promoter. True leaves of epidermal cells and mesophyll cells were observed 11 days after sowing. Bars, 10 µm. b Multimerization of cTP-mCherry in the transgenic plants. Immunoblot analysis of total proteins extracted from three lines each of cTP-sfGFP- or cTP-mCherry-expressing Arabidopsis leaves using Laemmli sample buffer with or without the reducing agent dithiothreitol (DTT). Samples extracted with the sample buffer without DTT were analyzed without boiling. Coomassie brilliant blue (CBB) staining shows blots of Rubisco large subunits (RbcL) as a loading control. Red dots, non-denatured form; red asterisks, denatured form; red line, multimers. c, d Comparison of cTP-fluorescent protein accumulation between cTP-sfGFP- and cTP-mCherry-expressing Arabidopsis plants. c Immunoblot analysis of total protein extracted from shoots of three lines each of Arabidopsis expressing cTP-sfGFP or cTP-mCherry. Total protein extracted from N. benthamiana leaves transiently expressing mCherry-sfGFP and its serial fivefold dilutions were used to compare antibody titers. CBB staining shows blots of RbcL as a loading control. d Quantification of cTP-fluorescent protein accumulation based on the immunoblot analysis in c. Different titers of anti-GFP and anti-RFP were adjusted by reactivity to mCherry-sfGFP. Accumulation levels in cTP-sfGFP #1 were set to 1.0. n = 3; data are shown as means ± SD; different letters indicate significant differences, P < 0.05, using Tukey’s multiple comparisons test. e, f Growth of Arabidopsis plants expressing cTP-sfGFP or cTP-mCherry. e Thirteen-day-old cTP-sfGFP- or cTP-mCherry-expressing Arabidopsis plants. Bars, 10 mm. f Quantification of plant length including roots and shoots. No significant difference was found in Tukey’s multiple comparison test. n = 23–31; data are shown as box and whisker plot; P < 0.05, using Tukey’s multiple comparisons test
Chloroplast–chloroplast adhesion impacts photorespiration-related pathways. a Heat map of metabolite accumulation in cTP-mCherry lines #1–3 (chloroplast-glued plants) and cTP-sfGFP lines #1–3 (transgenic control plants). Metabolites are listed in ascending order of P value for the difference between cTP-mCherry and cTP-sfGFP lines. The yellow square indicates metabolites with P < 0.05. Metabolites involved in the photorespiratory pathway are marked by asterisks. b, c Pathway enrichment analysis of metabolites whose accumulation significantly increased in chloroplast-glued plants. b Bubble chart. Higher-impacted metabolic pathways are indicated by larger red circles. c List of higher-impacted metabolic pathways. p, P values were calculated by Fisher’s exact test. FDR, the adjusted P value using the false discovery rate. Impact, the pathway impact value calculated from pathway topology analysis. Metabolic pathways with P < 0.05 and impact > 0 are listed in the table
Chloroplast–chloroplast adhesion causes accumulation of photorespiratory metabolites transported between organelles. a Simplified illustration of the transport of metabolites in the photorespiration pathway between organelles. b Quantification of the amount of glycine, glycerate, and serine in the wild-type (WT, non-transgenic plants), cTP-sfGFP-expressing lines #1–3 (control plants), or cTP-mCherry-expressing lines #1–3 (chloroplast-glued plants). Accumulation levels in the WT were set to 1.0. n = 7; data are shown as means ± SD; asterisks indicate significant differences between the WT, cTP-sfGFP-expressing lines, and cTP-mCherry-expressing lines, P < 0.05, using Dunnett’s multiple comparison test
Key message By using the organelle glue technique, we artificially manipulated organelle interactions and controlled the plant metabolome at the pathway level. Abstract Plant cell metabolic activity changes with fluctuating environmental conditions, in part via adjustments in the arrangement and interaction of organelles. This hints at the potential for designing plants with desirable metabolic activities for food and pharmaceutical industries by artificially controlling the interaction of organelles through genetic modification. We previously developed a method called the organelle glue technique, in which chloroplast–chloroplast adhesion is induced in plant cells using the multimerization properties of split fluorescent proteins. Here, we generated transgenic Arabidopsis (Arabidopsis thaliana) plants in which chloroplasts adhere to each other and performed metabolome analysis to examine the metabolic changes in these lines. In plant cells expressing a construct encoding the red fluorescent protein mCherry targeted to the chloroplast outer envelope by fusion with a signal sequence (cTP-mCherry), chloroplasts adhered to each other and formed chloroplast aggregations. Mitochondria and peroxisomes were embedded in the aggregates, suggesting that normal interactions between chloroplasts and these organelles were also affected. Metabolome analysis of the cTP-mCherry-expressing Arabidopsis shoots revealed significantly higher levels of glycine, serine, and glycerate compared to control plants. Notably, these are photorespiratory metabolites that are normally transported between chloroplasts, mitochondria, and peroxisomes. Together, our data indicate that chloroplast–chloroplast adhesion alters organellar interactions with mitochondria and peroxisomes and disrupts photorespiratory metabolite transport. These results highlight the possibility of controlling plant metabolism at the pathway level by manipulating organelle interactions.
Key message GRF–GIF chimeric proteins from multiple source species enhance in vitro regeneration in both wild and cultivated lettuce. In addition, they enhance regeneration in multiple types of lettuce including butterheads, romaines, and crispheads . Abstract The ability of plants to regenerate in vitro has been exploited for use in tissue culture systems for plant propagation, plant transformation, and genome editing. The success of in vitro regeneration is often genotype dependent and continues to be a bottleneck for Agrobacterium -mediated transformation and its deployment for improvement of some crop species. Manipulation of transcription factors that play key roles in plant development such as BABY BOOM, WUSCHEL, and GROWTH-REGULATING FACTORs (GRFs) has improved regeneration and transformation efficiencies in several plant species. Here, we compare the efficacy of GRF–GIF gene fusions from multiple species to boost regeneration efficiency and shooting frequency in four genotypes of wild and cultivated lettuce ( Lactuca spp. L.). In addition, we show that GRF–GIFs with mutated miRNA 396 binding sites increase regeneration efficiency and shooting frequency when compared to controls. We also present a co-transformation strategy for increased transformation efficiency and recovery of transgenic plants harboring a gene of interest. This strategy will enhance the recovery of transgenic plants of other lettuce genotypes and likely other crops in the Compositae family.
Key message Ectopic expression of MmCYP1A1 gene from Mus musculus in apple calli and Arabidopsis increased the levels of melatonin and 6-hydroxymelatonin, and improved their stress resistance. Abstract Melatonin occurs widely in organisms, playing a key regulatory role. CYP1A1 is a cytochrome P450 monooxygenase, involved in the melatonin metabolism, and is responsible for the synthesis of 6-hydroxymelatonin from melatonin. Melatonin and 6-hydroxymelatonin have strong antioxidant activities in animals. Here, we cloned MmCYP1A1 from Mus musculus and found that ectopic expression of MmCYP1A1 improved the levels of melatonin and 6-hydroxymelatonin in transgenic apple calli and Arabidopsis. Subsequently, we observed that MmCYP1A1 increased the tolerance of transgenic apple calli and Arabidopsis to osmotic stress simulated by polyethylene glycol 6000 (PEG 6000), as well as resistance of transgenic Arabidopsis to drought stress. Further, the number of lateral roots of MmCYP1A1 transgenic Arabidopsis were enhanced significantly after PEG 6000 treatment. The expression of MmCYP1A1 remarkably reduced malondialdehyde (MDA) content, electrolyte leakage, accumulation of H2O2 and O2⁻ during stress treatment. Moreover, MmCYP1A1 enhanced stress tolerance in apple calli and Arabidopsis by increasing the expression levels of resistance genes. MmCYP1A1 also promoted stomatal closure in transgenic Arabidopsis to reduce leaf water loss during drought. Our results indicate that MmCYP1A1 plays a key role in plant stress tolerance, which may provide a reference for future plant stress tolerance studies.
Key message Ectopic expression of PhAN2 in vegetative tissue can improve regeneration and adventitious rooting but inhibit axillary bud outgrowth of petunia, while overexpression specifically in flowers could shorten longevity. Abstract Anthocyanin 2 has been only treated as a critical positive regulation factor of anthocyanin biosynthesis in petunia flowers. To determine if this gene had other functions in plant growth, we overexpressed this gene in an an2 mutant petunia cultivar driven by promoters with different strengths or tissue specificity. Various physiological processes of transformants in different growth stages and environments were analyzed. Besides the expected pigmentation improvement in different tissues, the results also showed that ectopic expression of AN2 could improve the regeneration skill but inhibit the axillary bud germination of in vitro plants. Moreover, the rooting ability of shoot tips of transformants was significantly improved, while some transgenic lines’ flower longevity was shortened. Gene expression analysis showed that the transcripts level of AN2, partner genes anthocyanin 1 (AN1), anthocyanin 11 (AN11), and target gene dihydroflavonol 4-reductase (DFR) was altered in the different transgenic lines. In addition, ethylene biosynthesis-related genes 1-aminocyclopropane-1-carboxylic acid synthase (ACS1) and ACC oxidase (ACO1) were upregulated in rooting and flower senescence processes but at different time points. Overall, our data demonstrate that the critical role of this AN2 gene in plant growth physiology may extend beyond that of a single activator of anthocyanin biosynthesis.
Conceptual model of AUR1-TPX-MAP645 module controlling infection thread formation for legume nodulation. AUR1 interacts with MAP65-1/9, which leads to the phosphorylation of MAP65-1/9 via AUR1 in the presence of TPXL2/3. MYB3R1 binds over the MSA (mitosis-specific activator) elements present over the promoter of AUR1 for induction of AUR1 for infection thread formation
Key message We highlight the newly emerged regulatory role of a mitotic kinase AUR1, its activator, and its microtubule-associated proteins (MAPs) in infection thread formation for root nodule symbiosis.
Key message GhSCL13-2A, a member of the PAT1 subfamily in the GRAS family, positively regulates cotton resistance to Verticillium dahliae by mediating the jasmonic acid and salicylic acid signaling pathways and accumulation of reactive oxygen species. Abstract Verticillium wilt (VW) is a devastating disease of upland cotton (Gossypium hirsutum) that is primarily caused by the soil-borne fungus Verticillium dahliae. Scarecrow-like (SCL) proteins are known to be involved in plant abiotic and biotic stress responses, but their roles in cotton defense responses are still unclear. In this study, a total of 25 GhPAT1 subfamily members in the GRAS family were identified in upland cotton. Gene organization and protein domain analysis showed that GhPAT1 members were highly conserved. GhPAT1 genes were widely expressed in various tissues and at multiple developmental stages, and they were responsive to jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) signals. Furthermore, GhSCL13-2A was induced by V. dahliae infection. V. dahliae resistance was enhanced in Arabidopsis thaliana by ectopic overexpression of GhSCL13-2A, whereas cotton GhSCL13-2A knockdowns showed increased susceptibility. Levels of reactive oxygen species (ROS) and JA were also increased and SA content was decreased in GhSCL13-2A knockdowns. At the gene expression level, PR genes and SA signaling marker genes were down-regulated and JA signaling marker genes were upregulated in GhSCL13-2A knockdowns. GhSCL13-2A was shown to be localized to the cell membrane and the nucleus. Yeast two-hybrid and luciferase complementation assays indicated that GhSCL13-2A interacted with GhERF5. In Arabidopsis, V. dahliae resistance was enhanced by GhERF5 overexpression; in cotton, resistance was reduced in GhERF5 knockdowns. This study revealed a positive role of GhSCL13-2A in V. dahliae resistance, establishing it as a strong candidate gene for future breeding of V. dahliae-resistant cotton cultivars.
Key message The genomic location and stage-specific expression pattern of many long non-coding RNAs reveal their critical role in regulating protein-coding genes crucial in pollen developmental progression and male germ line specification. Abstract Long non-coding RNAs (lncRNAs) are transcripts longer than 200 bp with no apparent protein-coding potential. Multiple investigations have revealed high expression of lncRNAs in plant reproductive organs in a cell and tissue-specific manner. However, their potential role as essential regulators of molecular processes involved in sexual reproduction remains largely unexplored. We have used developing field mustard (Brassica rapa) pollen as a model system for investigating the potential role of lncRNAs in reproductive development. Reference-based transcriptome assembly performed to update the existing genome annotation identified novel expressed protein-coding genes and long non-coding RNAs (lncRNAs), including 4347 long intergenic non-coding RNAs (lincRNAs, 1058 expressed) and 2,045 lncRNAs overlapping protein-coding genes on the opposite strand (lncNATs, 780 expressed). The analysis of expression profiles reveals that lncRNAs are significant and stage-specific contributors to the gene expression profile of developing pollen. Gene co-expression networks accompanied by genome location analysis identified 38 cis-acting lincRNA, 31 cis-acting lncNAT, 7 trans-acting lincRNA and 14 trans-acting lncNAT to be substantially co-expressed with target protein-coding genes involved in biological processes regulating pollen development and male lineage specification. These findings provide a foundation for future research aiming at developing strategies to employ lncRNAs as regulatory tools for gene expression control during reproductive development.
Key message Comparative transcriptome analysis of early fruits of long and round eggplants, SmOVATE5, is involved in regulating fruit development. Abstract Eggplant, a solanaceous crop that has undergone a long period of domestication, is one of the most important vegetables worldwide. The shape of its fruit is an important agronomic trait and consumers in different regions have different preferences. However, a limited understanding of the molecular mechanisms regulating fruit development and shape has hindered eggplant breeding. In this study, we performed morphological observations and transcriptome analysis of long- and round-fruited eggplant genotypes to understand the molecular regulation during the early development of different fruit shapes. Morphological studies revealed that the two varieties already exhibited distinctly different phenotypes at the initial stage of fruit development before flowering, with rapid fruit enlargement beginning on the sixth day after flowering. Comparative transcriptome analysis identified phytohormone-related genes that were significantly upregulated on the day of flowering, indicating they may be involved in regulating the initial stages of fruit development. Notably, SmARF1 showed a sustained upregulation pattern in both varieties, suggesting that it may promote eggplant fruit growth. In addition, several differentially expressed genes of the SUN, YABBY, and OVATE families are potentially involved in the regulation of fruit development or fruit shape. We demonstrated that the SmOVATE5 gene has a negative regulatory function suppressing plant growth and development. In conclusion, this study provides new insights into the molecular regulatory mechanisms of eggplant fruit development, and the genes identified may provide valuable references for different fruit shape breeding programs.
Key message VaSUS2 enhances cold tolerance of transgenic tomato and Arabidopsis by regulating sucrose metabolism and improving antioxidant enzymes activity. Abstract Sucrose synthetase (SUS) is a key enzyme of sugar metabolism, and plays an important role in response to abiotic stress in plant. However, the function of VaSUS2 remains unknown in cold tolerance. Here, the cloning and functional characterization of the plasma membrane-localized VaSUS2 gene isolated from Vitis amurensis was studied. The transcript level of VaSUS2 was up-regulated under cold stress in Vitis amurensis. Heterologous expression of VaSUS2 in tomato increased SUS activity, which promoted the accumulation of glucose and fructose under cold treatment. The transgenic tomato and Arabidopsis exhibited higher levels of antioxidant enzymes activity, lower relative electrolyte leakage (REL), malondialdehyde (MDA) and hydrogen peroxide (H2O2) content compared to wild type under cold stress. Importantly, the ability of scavenging reactive oxygen species (ROS) in transgenic plants was significantly improved. Moreover, yeast two-hybrid (Y2H) indicated that VaSnRK1 might be a potential interaction protein of VaSUS2. qRT-PCR showed that sucrose metabolism-related genes SlSUS, SlSPS and SlINV were significantly up-regulated in transgenic tomatoes. Meanwhile, the expression levels of antioxidant enzyme genes and cold-related genes CBF1, COR47 and ICE1 were up-regulated in transgenic plants. Taken together, these results suggested that VaSUS2 was involved in cold tolerance by increasing the levels of soluble sugars, improving the activity of antioxidant enzymes, and up-regulating the expression of cold-related genes in transgenic tomatoes and Arabidopsis.
Expression levels of BR biosynthetic genes in developing woodland strawberry fruits. Gene expression levels of FveDWF4 (a, c) and FveDWF (b, d). Developmental stage-dependent expression in whole fruit (a, b). Flower organ-specific expression (c, d). Expression was normalized to the level of the FveGAPDH gene. Error bars indicate mean ± standard error; n = 3. The developmental stages of woodland strawberry are as follows: GB green bud stage, WD white dome stage (petals are visible but there is no anthesis), 0 DAP 0 days after pollination, 2 DAP 2 days after pollination, SG “small green stage”, LG “large green stage”, W white stage (beginning of the ripening), P stage characterized by a white receptacle with red achene, R fully ripened stage
Effects of propiconazole on the development of woodland strawberry. a Leaf and b flower phenotypes of mock- (top left), 250 µM PCZ- (top middle left), PCZ and 1 µM BL- (top middle right), and BL-treated plants (top right). Chlorophyll content of the leaves (a, bottom, n = 3). Petal area of flowers (b, bottom, n ≥ 10). c The size of well-formed fruits (n ≥ 10). d The proportion of matured fruits (n = 4–6). e The number of flowers formed within a month (n = 6). Bars indicate mean ± standard error. f Images of mature fruits under each treatment. PCZ-treated plants produced both well-formed (top middle) and misshapen fruits (top right)
Effects of PCZ on ovule development and fertilization. a The sizes of the ovules in mock- and 250 µM PCZ-treated (with or without 1 µM BL) plants. The sample of the length (solid line) and the width (dashed line) of ovule was shown in the picture. b Images of the carpel of the plants under each treatment (right). c Pollen tube guidance of mock- and PCZ-treated (with or without BL) plants. More than 30 achenes from two DAP fruit were stained with aniline blue and observed. The numbers of carpels in which the pollen tube was attracted to the micropyle were counted. Black dots indicate the proportion of ovules in which the pollen tube was attracted to the micropyle in each fruit, and the crosses indicate the mean proportions of each treatment (n ≥ 9). d–f Pollen tube guidance in mock- (d), PCZ- (e), and PCZ plus BL-treated (f) plants. White arrowheads indicate the pollen tubes. Asterisks indicate the estimated location of the micropyle. Bar = 100 µm
Effect of PCZ on female gametophyte gene expression. Gene expression of FveEC1 (a marker for egg cells, Sprunck et al. 2012), FveCKI1 (a marker for endosperm, Hejátko et al. 2003), and FveMYB98 (a marker for synergid cells, Kasahara et al. 2005) in the carpels were analyzed using qRT-PCR. Data are the mean ± standard error (n = 3)
Effect of PCZ on female gametophyte development. Images of mature a and immature b female gametophytes. es: Mature embryo sac (indicated by dashed line, Dziadczyk et al. 2011; Hollender et al. 2012; Leszczuk et al. 2018). Asterisk indicates megaspore-like cell (Dziadczyk et al. 2011; Leszczuk et al. 2018). Bar = 200 µm. c The proportions of mature and immature female gametophytes in mock- and PCZ-treated plants. Black dots indicate the proportion of ovules in each fruit containing normally developed female gametophyte, and the crosses indicate the mean proportions of each treatment (n = 6, some dots are overlapped)
Key Message In woodland strawberry, a brassinosteroid biosynthesis inhibitor propiconazole induced typical brassinosteroid-deficient phenotypes and decreased female fertility due to attenuated female gametophyte development. Abstract Brassinosteroids (BRs) play roles in various aspects of plant development. We investigated the physiological roles of BRs in the woodland strawberry, Fragaria vesca. BR-level-dependent phenotypes were observed using a BR biosynthetic inhibitor, propiconazole (PCZ), and the most active natural BR, brassinolide (BL). Endogenous BL and castasterone, the active BRs, were below detectable levels in PCZ-treated woodland strawberry. The plants were typical BR-deficient phenotypes, and all phenotypes were restored by treatment with BL. These observations indicate that PCZ is an effective inhibitor of BR in woodland strawberry. Only one gene for each major step of BR biosynthesis in Arabidopsis is encoded in the woodland strawberry genome. BR biosynthetic genes are highly expressed during the early stage of fruit development. Emasculated flowers treated with BL failed to develop fruit, implying that BR is not involved in parthenocarpic fruit development. Similar to BR-deficient and BR-insensitive Arabidopsis mutants, female fertility was lower in PCZ-treated plants than in mock-treated plants due to failed attraction of the pollen tube to the ovule. In PCZ-treated plants, expression of FveMYB98, the homologous gene for Arabidopsis MYB98 (a marker for synergid cells), was downregulated. Ovules were smaller in PCZ-treated plants than in mock-treated plants, and histological analysis implied that the development of more than half of female gametophytes was arrested at the early stage in PCZ-treated plants. Our findings explain how BRs function during female gametophyte development in woodland strawberry.
Key message A highly efficient transformation procedure to generate transgenic Stylosanthes roots was established. SgEXPB1 is involved in Stylosanthes root growth under phosphorus deficiency. Abstract Stylo (Stylosanthes spp.) is an important forage legume widely applied in agricultural systems in the tropics. Due to the recalcitrance of stylo genetic transformation, functional characterization of candidate genes involved in stylo root growth is limited. This study established an efficient procedure for Agrobacterium rhizogenes-mediated transformation for generating transgenic composite plants of S. guianensis cultivar ‘Reyan No. 5’. Results showed that composite stylo plants with transgenic hairy roots were efficiently generated by A. rhizogenes strains K599 and Arqual, infecting the residual hypocotyl at 1.0 cm of length below the cotyledon leaves of 9-d-old seedlings, leading to a high transformation efficiency of > 95% based on histochemical β-glucuronidase (GUS) staining. Notably, 100% of GUS staining-positive hairy roots can be achieved per composite stylo plant. Subsequently, SgEXPB1, a β-expansin gene up-regulated by phosphorus (P) deficiency in stylo roots, was successfully overexpressed in hairy roots. Analysis of hairy roots showed that root growth and P concentration in the transgenic composite plants were increased by SgEXPB1 overexpression under low-P treatment. Taken together, a highly efficient A. rhizogenes-mediated transformation procedure for generating composite stylo plants was established to study the function of SgEXPB1, revealing that this gene is involved in stylo root growth during P deficiency.
Key message IiSVP of Isatis indigotica was cloned and its expression pattern was analyzed. Ectopic expression of IiSVP in Arabidopsis could delay the flowering time and reduce the size of the floral organs. Abstract SVP (SHORT VEGETATIVE PHASE) can negatively regulate the flowering time of Arabidopsis. In the present work, the cDNA of IiSVP, an orthologous gene of AtSVP in I. indigotica, was cloned. IiSVP was highly expressed in rosette leaves, inflorescences and petals, but weakly expressed in sepals, pistils and young silicles. The results of subcellular localization showed that IiSVP was localized in nucleus. Bioinformatics analysis indicated that this protein was a MADS-box transcription factor. Constitutive expression of IiSVP in Arabidopsis thaliana resulted in decrease of the number of petals and stamens, and curly sepals were formed. In IiSVP transgenic Arabidopsis plants, obvious phenotypic variations in flowers could be observed, especially the size of the floral organs. In comparison with the wild-type plants, the size of petals, stamens and pistil in IiSVP transgenic Arabidopsis plants was decreased significantly. In some transgenic plants, the petals were wrapped by the sepals. Yeast two-hybrid experiments showed that IiSVP could form higher-order complexes with other MADS proteins, including IiSEP1, IiSEP3, IiAP1 and IiSEP4, but could not interact with IiSEP2. In this work, it was proved that the flowering process and the floral development in Arabidopsis could be affected by IiSVP from I. indigotica Fortune.
Key message We extended the applicability of the BY-2 cell line as a model by introducing two new selection systems. Our protocol provides guidelines for optimising Basta selection in other recalcitrant models. Abstract Tobacco BY-2 cell line is the most commonly used cytological model in plant research. It is uniform, can be simply treated by chemicals, synchronised and easily transformed. However, only a few selection systems are available that complicate advanced studies using multiple stacked transgenes and extensive gene editing. In our work, we adopted for BY-2 cell line two other selection systems: sulfadiazine and phosphinothricin (PPT, an active ingredient of Basta herbicide). We show that sulfadiazine can be used in a wide range of concentrations. It is suitable for co-transformation and subsequent double selection with kanamycin or hygromycin, which are standardly used for BY-2 transformation. We also have domesticated the sulfadiazine resistance for the user-friendly GoldenBraid cloning system. Compared to sulfadiazine, establishing selection on phosphinothricin was considerably more challenging. It did not work in any concentration of PPT with standardly cultured cells. Since the selection is based on blocking glutamine synthetase and consequent ammonium toxicity and deficiency of assimilated nitrogen, we tried to manipulate nitrogen availability. We found that the PPT selection reliably works only with nitrogen-starved cells with reduced nitrate reserves that are selected on a medium without ammonium nitrate. Both these adjustments prevent the release of large amounts of ammonium, which can toxify the entire culture in the case of standardly cultured cells. Since high nitrogen reserves can be a common feature of in vitro cultures grown on MS media, nitrogen starvation could be a key step in establishing phosphinothricin resistance in other plant models.
Key message Arabidopsis nucleoporin involved in the regulation of ethylene signaling via controlling of nucleocytoplasmic transport of mRNAs. AbstractThe two-way transport of mRNAs between the nucleus and cytoplasm are controlled by the nuclear pore complex (NPC). In higher plants, the NPC contains at least 30 nucleoporins. The Arabidopsis nucleoporins are involved in various biological processes such as pathogen interaction, nodulation, cold response, flowering, and hormone signaling. However, little is known about the regulatory functions of the nucleoporin NUP160 and NUP96 in ethylene signaling pathway. In the present study, we provided data showing that the Arabidopsis nucleoporin NUP160 and NUP96 participate in ethylene signaling-related mRNAs nucleocytoplasmic transport. The Arabidopsis nucleoporin mutants (nup160, nup96-1, nup96-2) exhibited enhanced ethylene sensitivity. Nuclear qRT-PCR analysis and poly(A)-mRNA in situ hybridization showed that the nucleoporin mutants affected the nucleocytoplasmic transport of all the examined mRNAs, including the ethylene signaling-related mRNAs such as ETR2, ERS1, ERS2, EIN4, CTR1, EIN2, and EIN3. Transcriptome analysis of the nucleoporin mutants provided clues suggesting that the nucleoporin NUP160 and NUP96 may participate in ethylene signaling via various molecular mechanisms. These observations significantly advance our understanding of the regulatory mechanisms of nucleoporin proteins in ethylene signaling and ethylene response.
Key message Eleven Alfin-like (AL) genes were obtained from apple and MdAL4 was selected for improving drought stress tolerance of transgenic apple callus and Arabidopsis. Abstract Drought is an important environmental factor affecting plant growth all over the world. Alfin-like (AL) have well-documented functions in abiotic stress response, but their drought stress tolerance in apple (Malus domestica) are poorly understood. According to the transcriptome data, 11 MdAL genes containing conserved Alfin and PHD-finger domain were identified in apple and divided into three subgroups with a total of 35 members from different species. Subsequently, gene structures, conserved amino acid sequences, promoter cis-acting elements, and gene evolution events were analyzed. Based on differential expression of MdALs in response to abiotic stresses, MdAL4, which was highly expressed under drought, was further cloned and investigated. MdAL4 encoding nuclear-localized protein conferred enhanced drought tolerance in overexpressing transgenic calli of apple ‘Orin’. Moreover, the ectopic expression of MdAL4 improved the drought tolerance of transgenic Arabidopsis, as judged from remarkably decreased malonaldehyde (MDA) content and electrolyte leakage in MdAL4 overexpressing plants relative to WT. Furthermore, MdAL4 possibly could bind to promoter regions of ROS-scavenging and stress-related genes to improve drought tolerance. Additionally, we found in silico evidence that three proteins containing the WD40 domain that interact with MdAL4. Based on these results, MdAL4 was identified as a positive regulator for improving drought stress of apple.
Key message WSL214 plays an important role in promoting cellular ROS homeostasis by enhancing catalase activity and reducing photosynthetic ROS production. AbstractROS are the important regulator of cellular homeostasis, and balancing ROS production and clearance contributes to cellular activity. Although many genes associated with ROS have been cloned, the mechanism of this balance is not fully understood. In this study, we obtained the rice mutant wsl214 that arose from a natural mutation. Compared to WT, wsl214 exhibited white-striped leaves, defective chloroplast development, reduced net photosynthetic rate, and overexcitation of photosynthetically active reaction centers. In addition, the ROS accumulation level was significantly elevated, and the ROS scavenging enzyme activity was significantly decreased in wsl214 leaf tissue. As a result of elevated ROS levels, wsl214 leaf cells underwent DNA damage and programmed cell death. However, wsl214 defense response to exogenous pathogens was also enhanced by high ROS levels. Based on the mapping cloning, we discovered that WSL214 had a single base mutation (C to T) in the third exon, resulting in decreased expression of wsl214. The WSL214 encodes an HD domain phosphohydrolase and is widely expressed in various tissues of rice, especially at the highest level in leaf tissue. Further research showed that WSL214 promoted the homeostasis of rice leaf cellular ROS in two ways. First, WSL214 increased the expression of the catalase gene OsCATC, making the intracellular ROS scavenging enzyme more active. Second, WSL214 promoted chloroplast development, kept photosynthesis working properly, and reduced ROS produced by photosynthesis. In conclusion, our report emphasizes that WSL214 is a key part of balancing ROS levels in cells.
Key message Overexpression in Arabidopsis of the maize shikimate kinase-like genes SKL1 and SKL2 enhances tolerance to drought stress. Abstract The shikimate pathway has been reported to play an important role in plant signaling, reproduction, and development. However, its role in abiotic stress has not yet been reported. Here, two shikimate kinase-like genes, SKL1 and SKL2, were cloned from maize and their functions in mediating drought tolerance were investigated. Transcript levels of ZmSKL1 and ZmSKL2 in roots and leaves were strongly induced by drought stress. Both proteins were localized in the chloroplast. Furthermore, compared to the wild-type, transgenic Arabidopsis plants overexpressing ZmSKL1 or ZmSKL2 exhibited improved drought stress tolerance through increases in relative water content and stomatal closure. Additionally, the transgenic lines showed reduced accumulation of reactive oxygen species as a results of increased antioxidant enzyme activity. Interestingly, overexpression of ZmSKL1 or ZmSKL2 also increased sensitivity to exogenous abscisic acid. In addition, the ROS-related and stress-responsive genes were activated in transgenic lines under drought stress. Moreover, ZmSKL1 and ZmSKL2 were found to separately interact with ZmASR3, which is an important regulatory protein in mediating drought tolerance, suggesting that ZmSKL1 and ZmSKL2, together with ZmASR3, are proteins that may confer drought tolerance as candidates in plant genetic breeding manipulations.
Key message OsPPR11 belongs to the P-type PPR protein family and can interact with OsCAF2 to regulate Group II intron splicing and affect chloroplast development in rice. Abstract Pentatricopeptide repeat (PPR) proteins participate in chloroplasts or mitochondria group II introns splicing in plants. The PPR protein family contains 491 members in rice, but most of their functions are unknown. In this study, we identified a nuclear gene encoding the P-type PPR protein OsPPR11 in chloroplasts. The qRT-PCR analysis demonstrated that OsPPR11 was expressed in all plant tissues, but leaves had the highest expression. The osppr11 mutants had yellowing leaves and a lethal phenotype that inhibited chloroplast development and photosynthesis-related gene expression and reduced photosynthesis-related protein accumulation in seedlings. Moreover, photosynthetic complex accumulation decreased significantly in osppr11 mutants. The OsPPR11 is required for ndhA, and ycf3-1 introns splicing and interact with CRM family protein OsCAF2, suggesting that these two proteins may form splicing complexes to regulate group II introns splicing. Further analysis revealed that OsCAF2 interacts with OsPPR11 through the N-terminus. These results indicate that OsPPR11 is essential for chloroplast development and function by affecting group II intron splicing in rice.
Key message Novel function and mechanism of a PNP molecule VaEG45 from adzuki bean involved in plant immunity. Abstract Plant natriuretic peptides (PNPs) can affect a broad spectrum of physiological responses in plants acting as peptidic signaling molecules. However, PNPs may play additional roles in plant immunity. Our previous transcriptome data of adzuki bean (Vigna angularis) in response to Uromyces vignae infection revealed association of PNP-encoding gene VaEG45 with U. vignae resistance. To determine the function of VaEG45 in disease resistance, we cloned the 589 bp nucleotide sequence of VaEG45 containing 2 introns, encoding a putative 13.68 kDa protein that is 131 amino acids in length. We analyzed expression in different resistant cultivars of V. angularis and found significant induction of VaEG45 expression after U. vignae infection. Transient expression of VaEG45 improved tobacco resistance against Botrytis cinerea. We next analyzed the mechanism by which VaEG45 protects plants from fungal infection by determination of the biological activity of the prokaryotic expressed VaEG45. The results showed that the fusion protein VaEG45 can significantly inhibit urediospores germination of U. vignae, mycelial growth, and the infection of tobacco by B. cinerea. Further analysis revealed that VaEG45 exhibits β-1, 3-glucanase activity. These findings uncover the function of a novel PNP molecule VaEG45 and provide new evidence about the mechanism of PNPs in plant immunity.
Dynamically reversible processes of ubiquitination and deubiquitination. The red star pattern at the center of DUB representing the activated catalytic site. The cascade reaction of ubiquitination is an ATP-consuming process. Ub is first activated by a Ub-activating enzyme (Uba or E1), and the activated Ub can be transferred to the active moiety of Ub-conjugating enzyme (Ubc or E2) to form a high-energy thioester intermediate Ub ~ E2. In the presence of Ub ligase (E3), the Ub is then transferred from E2 ~ Ub to a surface Lys residue of the target protein. In order to keep a balance of the entire Ub pool, deubiquitinases (DUBs) can process the existing mono-Ub or poly-Ub chains on the target substrates. The S1 site of DUBs helps the formation of enzyme–substrate complex. In many cases, the S1 site is occupied by the distal Ub, whereas the S1’ site is occupied by the proximal Ub or by specific substrates. The catalytic site of DUBs will break linkages between Ub molecules or between Ub and the substrate. After that, all these modules will be recycled for another round, while the substrate whose fate is being digested will be degraded by 26S proteasome
Phylogenetic tree of seven DUB subfamilies in Arabidopsis thaliana and their candidate homologs in Zea mays, Oryza sativa, and Triticum aestivum. All the candidate homologs are found by using the TBtools software (Chen et al. 2020). The phylogenetic tree was constructed by using MEGA7.0 software by the maximum-likelihood method with 1,000 bootstrap replicates. Additionally, the modification process of this phylogenetic tree is accomplished on the iTOL website: Different colors of branches and chunks indicate distinct DUB subfamilies as well as four diverse species; bootstrap values are shown on each branch as circles
Schematic of the domain structures of HsAtaxin3, At1g07300, At2g29640, and At3g54130. The Cys residue in active site 1 (black asterisk) is predicted to be a catalytic site identified in most thiol-dependent cysteine-type proteases. The His and Leu residues (red arrow) in active sites 2 and 3, respectively, are predicted as the catalytic sites by Uniprot online database ( However, there is no conserved active sites within At1g07300. UIM Ub-interacting motif (colour figure online)
Conserved domain alignment between HsZUFSP and candidate members in the Arabidopsis ZUFSP subfamily, including AT5G24680 and AT3G48380. HsZUFSP functional domains are based on Haahr et al. (2018). The black asterisk marks the catalytic Cys, red arrows mark catalytic His residues and the green arrow notes the catalytic His residue of UFM1 (ubiquitin-fold modifier1)-specific proteases. Although the ZUFSP peptidase domain shows similarity to established UFM1-specific proteases, it lacks the catalytic His residue found in these proteins and is therefore thought to be catalytically inactive. Conserved residues in the UBZ and MIU motifs are marked by red asterisks (colour figure online)
Putative Arabidopsis MINDY proteins in comparison to human homologs. HsMINDY1 is aligned with its close homologs, At4g11860 and At4g22960 (upper panel), while HsMINDY3 is aligned with its close homolog At1g43690 (lower panel). The domain and motif prediction were performed by the CDD and MEME databases linked by TBtools (Chen et al. 2020). Black asterisks indicate predicted catalytic residues, red arrows indicate conserved residues in the Cys loop. In addition, the InterPro online database (0 predicts that At1g43690 contains a MIU at its N-terminus (colour figure online)
Key message This article attempts to provide comprehensive review of plant deubiquitinases, paying special attention to recent advances in their biochemical activities and biological functions. Abstract Proteins in eukaryotes are subjected to post-translational modifications, in which ubiquitination is regarded as a reversible process. Cellular deubiquitinases (DUBs) are a key component of the ubiquitin (Ub)–proteasome system responsible for cellular protein homeostasis. DUBs recycle Ub by hydrolyzing poly-Ub chains on target proteins, and maintain a balance of the cellular Ub pool. In addition, some DUBs prefer to cleave poly-Ub chains not linked through the conventional K48 residue, which often alter the substrate activity instead of its stability. In plants, all seven known DUB subfamilies have been identified, namely Ub-binding protease/Ub-specific protease (UBP/USP), Ub C-terminal hydrolase (UCH), Machado–Joseph domain-containing protease (MJD), ovarian-tumor domain-containing protease (OTU), zinc finger with UFM1-specific peptidase domain protease (ZUFSP), motif interacting with Ub-containing novel DUB family (MINDY), and JAB1/MPN/MOV34 protease (JAMM). This review focuses on recent advances in the structure, activity, and biological functions of plant DUBs, particularly in the model plant Arabidopsis.
Key message Overexpression of the Aux/IAA protein TaIAA15-1A from wheat improves drought tolerance by regulating the ABA signalling pathway in transgenic Brachypodium. Abstract Drought is a major abiotic stress that causes severe crop yield loss. Aux/IAA genes have been shown to be involved in drought stress responses. However, to the best of our knowledge, there has been little research on the molecular mechanism of the wheat Aux/IAA gene in the context of drought tolerance. In this study, we found that expression of the wheat Aux/IAA gene TaIAA15-1A was upregulated by PEG6000, NaCl, SA, JA, IAA and ABA. Transgenic plants overexpressing TaIAA15-1A showed higher drought tolerance than wild-type (WT) plants. The physiological analyses showed that the transgenic lines exhibited a higher survival rate, shoot length, and relative water content than the WT plants. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were enhanced in transgenic lines, causing a reduction in the hydrogen peroxide (H2O2) and superoxide anion radical (O2⁻) contents. Transcriptome analysis showed that TaIAA15-1A overexpression alters the expression of these genes involved in the auxin signalling pathway, ABA signalling pathway, phenolamides and antioxidant pathways. The results of exogenous ABA treatment suggested that TaIAA15-1A overexpression increased sensitivity to ABA at the germination and postgermination stages compared to WT plants. These results indicate that TaIAA15-1A plays a positive role in plant drought tolerance by regulating ABA-related genes and improving antioxidative stress ability and has potential application in genetically modified crops.
Key message Auxin accumulation upregulates the expression of APETALA1 (CmAP1) and subsequently activates inflorescence primordium development in axillary buds of chestnut. Abstract The architecture of fruiting branches is a key determinant of chestnut yield. Normally, axillary buds at the top of mother fruiting branches develop into flowering shoots and bear fruits, and the lower axillary buds develop into vegetative shoots. Decapitation of the upper axillary buds induces the lower buds to develop into flowering shoots. How decapitation modulates the tradeoff between vegetative and reproductive development is unclear. We detected inflorescence primordia within both upper and lower axillary buds on mother fruiting branches. The level of the phytohormones 3-indoleacetic acid (IAA) and trans-zeatin (tZ) increased in the lower axillary buds in response to decapitation. Exogenous application of the synthetic analogues 1-naphthylacetic acid (NAA) or 6-benzyladenine (6-BA) blocked or promoted, respectively, the development of the inflorescence primordia in axillary buds. The transcript levels of the floral identity gene CmAP1 increased in axillary buds following decapitation. An auxin response element TGA-box is present in the CmAP1 promoter and influenced the CmAP1 promoter-driven expression of β-glucuronidase (GUS) in floral organs in Arabidopsis, suggesting that CmAP1 is induced by auxin. We propose that decapitation releases axillary bud outgrowth from inhibition caused by apical dominance. During this process, decapitation-induced accumulation of auxin induces CmAP1 expression, subsequently promoting the reproductive development of axillary buds.
Key message Our findings indicated that the SlERF.J2-IAA23 module integrates hormonal signals to regulate hypocotyl elongation and plant height in tomato. AbstractLight and phytohormones can synergistically regulate photomorphogenesis-related hypocotyl elongation and plant height in tomato. AP2/ERF family genes have been extensively demonstrated to play a role in light signaling and various hormones. In this study, we identified a novel AP2/ERF family gene in tomato, SlERF.J2. Overexpression of SlERF.J2 inhibits hypocotyl elongation and plant height. However, the plant height in the slerf.j2ko knockout mutant was not significantly changed compared with the WT. we found that hypocotyl cell elongation and plant height were regulated by a network involving light, auxin and gibberellin signaling, which is mediated by regulatory relationship between SlERF.J2 and IAA23. SlERF.J2 protein could bind to IAA23 promoter and inhibit its expression. In addition, light–dark alternation can activate the transcription of SlERF.J2 and promote the function of SlERF.J2 in photomorphogenesis. Our findings indicated that the SlERF.J2-IAA23 module integrates hormonal signals to regulate hypocotyl elongation and plant height in tomato.
Key message R2R3 MYB transcription factor GhMYB18 is involved in the defense response to cotton aphid by participating in the synthesis of salicylic acid and flavonoids. Abstract R2R3 MYB transcription factors (TFs) play crucial roles in plant growth and development as well as response to abiotic and biotic stresses. However, the mechanism of R2R3 MYB TFs in cotton response to aphid infestation remains largely unknown. Here, an R2R3 MYB transcription factor GhMYB18 was identified as a gene up-regulated from upland cotton (Gossypium hirsutum L.) under cotton aphid (Aphis gossypii Glover) infestation. GhMYB18, which has transcription activity, was localized mainly to nucleus and cell membranes. Transient overexpression of GhMYB18 in cotton activates salicylic acid (SA) and phenylpropane signaling pathways and promoted the synthesis of salicylic acid and flavonoids, which leads to enhancing the tolerance to cotton aphid feeding. In contrast, silencing of GhMYB18 increased the susceptibility of G. hirsutum to aphid. Additionally, GhMYB18 significantly promoted the activities of defense-related enzymes including catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL). These results collectively suggest that GhMYB18 is involved in cotton defense response to cotton aphid attacks through regulating the synthesis of salicylic acid and flavonoids.
Effect of in vitro RNAi of target genes and their fusion cassettes on transcripts abundance of corresponding genes in Meloidogyne incognita J2s. a J2s silenced for individual target genes, Mi-msp1, Mi-msp9, Mi-msp16, Mi-msp40, Mi-pel, Mi-pel2, Mi-pel3, Mi-xyl2, Mi-pg, and Mi-sp of M. incognita; b Fusion cassette-1 having Mi-msp1, Mi-msp9, Mi-msp16, Mi-msp40;c Fusion cassette-2 having Mi-pel, Mi-pel2, Mi-pel3, Mi-xyl2, Mi-pg, and Mi-sp. Gene expression (normalized with 18S rRNA and actin of M. incognita) was quantified by 2−ΔΔCT method. Each bar represents the log2-transformed mean of qRT-PCR runs in three biological and three technical replicates with standard errors. Asterisks indicate significant differential expression compared to that of J2s treated with gfp dsRNA and worms in water at P < 0.05 and P < 0.01
Quantification of transcripts abundance of key RNAi pathway genes involved in dsRNA processing in fusion-dsRNA silenced J2s of Meloidogyne incognita. 18 s rRNA and actin of M. incognita was used as a reference gene and fold-change was calculated using the 2−ΔΔCT method. Each bar represents the log2-transformed mean of qRT-PCR runs in three biological and three technical replicates with standard errors. Asterisks indicate significant differential expression compared to that of J2s treated with gfp dsRNA and water control at P < 0.05 and P < 0.01
Effect of in vitro RNAi of target genes, Mi-msp1, Mi-msp9, Mi-msp16, Mi-msp40, Mi-pel, Mi-pel2, Mi-pel3, Mi-xyl2, Mi-pg, and Mi-sp individually and their fusion genes (fusion cassette-1 and fusion cassette-2) on M. incognita J2s. a Nematode attraction at 4, 8 h showing J2s around the roots; b Nematode penetration in tomato roots on PF-127 medium at 24 and 72 h, cM. incognita development and reproduction in adzuki beans in the CYG pouches at 30 dpi. Each bar represents the mean ± standard error (n = 10), and bars with different letters indicate a significant difference at P < 0.05. Nematodes soaked with gfp dsRNA and worms in water were used as the controls
Molecular characterizations of transgenic plants. Southern hybridization of T1 events harboring the HD-RNAi constructs of a fusion cassette-1, and b fusion cassette-2; (lanes F1–E1 to F1–E8 and F2–E1 to F2–E8 shows different transgenic events); c, d Northern hybridization of T1 events harboring the HD-RNAi constructs of fusion cassette-1 and fusion cassette-2 for the presence and expression of small interfering RNAs (siRNAs); representative blots showing siRNAs (21–24 bp long) specific to Mi-msp16 in case of HD-RNAi events of fusion-1 and Mi-pg for fusion-2. P positive control, B blank, WT negative control (wild-type plant); e, f Detection of M. incognita target gene expression in T1 events by qRT-PCR. Relative transcript levels of Mi-msp1, Mi-msp9, Mi-msp16, Mi-msp40 in fusion-1 transformed events, and Mi-pel, Mi-pel2, Mi-pel3, Mi-xyl2, Mi-pg, Mi-sp in fusion-2 transformed events are expressed as Δct values which represent the difference in ct mean of transgene and the reference gene (N. tabacum 18S rRNA). Higher Δct values specify the lower expression of transgene in the corresponding event. Each bar indicates mean ± standard error determined from three independent biological and three technical replicates. Bars with different letters are significantly different at P < 0.05
HD-RNAi effect of fusion genes on development and reproduction of M. incognita in N. tabacum as well as on transcripts abundance of target genes. Performance of transgenic events was evaluated for nematode disease burden in terms of number of galls, total endoparasites, egg masses, eggs per egg mass, and nematode multiplication factor (MF) in different transgenic events of a fusion-1 (F1–E1 to F1–E8); b fusion-2 (F2–E1 to F2–E8), and WT plants at 35 dpi in soil. Each bar represents the mean ± standard error (n = 6), and bars with different letters indicate a significant difference at P < 0.05; c, d gravid females that developed were dissected out of the transgenic events showed comparatively smaller size (average length 690 ± 35 μm; width 500 ± 30 μm) than those from WT plants (average length 880 ± 40 μm; width 640 ± 50 μm). Each bar represents the mean ± SE of n = 10; bars with different letters denote a significant difference at P < 0.05. Scale bar = 500 μm; e, f Relative transcript levels of target transgenes in M. incognita females collected from dsRNA expressing transgenic events of fusion-1: F1–E1 to F1–E8, and fusion-2: F2–E1 to F2–E8. Gene expression normalized with 18S rRNA of M. incognita was quantified by 2−ΔΔCT method. Each bar represents the log2-transformed mean of qRT-PCR runs in three biological and three technical replicates with standard errors. Bars with different letters specify the significant differential expression at P < 0.05 in comparison with the females extracted from WT plants
Key message This study demonstrates multi-gene silencing approach for simultaneous silencing of several functional genes through a fusion gene strategy for protecting plants against root-knot nematode,Meloidogyne incognita. The ability of root-knot nematode (RKN), Meloidogyne incognita, to cause extensive yield decline in a wide range of cultivated crops is well-documented. Due to the inadequacies of current management approaches, the alternatively employed contemporary RNA interference (RNAi)-based host-delivered gene silencing (HD-RNAi) strategy targeting different functional effectors/genes has shown substantial potential to combat RKNs. In this direction, we have explored the possibility of simultaneous silencing of four esophageal gland genes, six plant cell-wall modifying enzymes (PCWMEs) and a serine protease gene of M. incognita using the fusion approach. In vitro RNAi showed that combinatorial gene silencing is the most effective in affecting nematode behavior in terms of reduced attraction, penetration, development, and reproduction in tomato and adzuki beans. In addition, qRT-PCR analysis of M. incognita J2s soaked in fusion-dsRNA showed perturbed expression of all the genes comprising the fusion construct confirming successful dsRNA processing which is also supported by increased mRNA abundance of five key-RNAi pathway genes. In addition, hairpin RNA expressing constructs of multi-gene fusion cassettes were developed and used for generation of Nicotiana tabacum transgenic plants. The integration of gene constructs and expression of siRNAs in transgenic events were confirmed by Southern and Northern blot analyses. Besides, bio-efficacy analyses of transgenic events, conferred up to 87% reduction in M. incognita multiplication. Correspondingly, reduced transcript accumulation of the target genes in the M. incognita females extracted from transgenic events confirmed successful gene silencing.
Phenylpropanoid biosynthetic pathway in hairy roots of Salvia miltiorrhiza. The heat maps showed the expression level of DEGs in Sm4CL2-OE-4 and anti-Sm4CL2-1 hairy roots. Red, up-regulated; blue, down-regulated. Different colors indicate different gene expression levels based on the log2FC values
Terpenoid metabolism pathway in hairy roots of Salvia miltiorrhiza. The heat maps showed the expression level of DEGs between anti-Sm4CL2-1 and control-3 hairy roots. Red, up-regulated; blue, down-regulated. Different colors represent different gene expression levels by the log2FC values
Morphology and typical cross section view of Sm4CL2-OE-4, anti-Sm4CL2-1 and control-3 hairy roots. a Hairy roots cultured in MSOH liquid medium for 21 days and MSOH solid medium for 6 weeks (photographed through the bottom of the flask). Red arrow indicates calli. b Typical cross-section view of Sm4CL2-OE, anti-Sm4CL2-1 and control-3 hairy roots
The top enriched KEGG pathways of the DEGs in danshen hairy roots. aSm4CL2-OE-4 vs control-3. b anti-Sm4CL2-1 vs control-3
Key message Overexpression and antisense expression of Sm4CL2 re-directed the biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots. Abstract Danshen (Salvia miltiorrhiza Bunge) is a widely used traditional Chinese medicine and its main active ingredients are water-soluble phenolic acids and lipophilic diterpenoids which are produced through the phenylpropanoid pathway and terpenoid pathway, respectively. 4-Coumaric acid: Coenzyme A ligase (4CL) is a key enzyme in the phenylpropanoid metabolism. We had obtained Sm4CL2-overexpressing (Sm4CL2-OE) and antisense Sm4CL2-expressing (anti-Sm4CL2) danshen hairy roots over ten years ago. In the follow-up study, we found that total salvianolic acids in Sm4CL2-OE-4 hairy roots increased to 1.35 times of the control-3, and that in anti-Sm4CL2-1 hairy roots decreased to 37.32% of the control-3, but tanshinones in anti-Sm4CL2-1 was accumulated to 1.77 ± 0.16 mg/g of dry weight, compared to undetectable in Sm4CL2-OE-4 and the control-3 hairy roots. Interestingly, Sm4CL2-OE-4 hairy roots contained more lignin, 1.36 times of the control-3, and enhanced cell wall and xylem lignification. Transcriptomic analysis revealed that overexpression of Sm4CL2 caused the upregulation of other phenylpropanoid pathway genes and antisense Sm4CL2 expression resulted in the downregulation of other phenylpropanoid pathway genes but activated the expression of terpenoid pathway genes like SmCYP76AK5, SmGPPS.SSUII.1 and SmDXS2. Protein–protein interaction analysis suggested that Sm4CL2 might interact with PAL, PAL4, CSE, CCoAOMT and SmCYP84A60, and appeared to play a key role in the interaction network. The tracking work in this study proved that Sm4CL2 could redirect both salvianolic acids and tanshinones biosynthesis possibly through synergistically regulating other pathway genes. It also indicated that genetic modification of plant secondary metabolism with biosynthetic gene might cause other responses through protein–protein interactions.
Key message OsJAZ2 protein has a propensity to form condensates, possibly by multivalent interactions, and can be used to construct artificial compartments in plant cells. Abstract Eukaryotic cells contain various membraneless organelles, which are compartments consisting of proteinaceous condensates formed by phase separation. Such compartments are attractive for bioengineering and synthetic biology, because they can modify cellular function by the enrichment of molecules of interest and providing an orthogonal reaction system. This study reports that Oryza sativa JAZ2 protein (OsJAZ2) is an atypical jasmonate signalling regulator that can form large condensates in both the nucleus and cytosol of O. sativa cells. TIFY and Jas domains and low-complexity regions contribute to JAZ2 condensation, possibly by multivalent interaction. Fluorescence recovery after photobleaching (FRAP) analysis suggests that JAZ2 condensates form mostly gel-like or solid compartments, but can also be in a liquid-like state. Deletion of the N-terminal region or the TIFY domain of JAZ2 causes an increase in the mobile fraction of JAZ2 condensates, moderately. Moreover, JAZ2 can also form liquid-like condensates when expressed in Nicotiana benthamiana cells. The recombinant JAZ2 fused to the green fluorescent protein (GFP) forms condensate in vitro, suggesting that the intermolecular interaction of JAZ2 molecules is a driving force for condensation. These results suggest the potential use of JAZ2 condensates to construct artificial membraneless organelles in plant cells.
Key message OsVP1 and Sdr4 play an important role in regulating seed dormancy that involved in multiple metabolism and regulatory pathways. Abstract Seed dormancy and germination are critical agricultural traits influencing rice grain yield. Although there are some genes have identified previously, the comprehensive understanding based on transcriptome is still deficient. In this study, we generated mutants of two representative regulators of seed germination, Oryza sativa Viviparous1 (OsVP1) and Seed dormancy 4 (Sdr4), by CRISPR/Cas9 approach and named them cr-osvp1 and cr-sdr4. The weakened dormancy of mutants indicated that the functions of OsVP1 and Sdr4 are required for normal early seed dormancy. There were 4157 and 8285 differentially expressed genes (DEGs) were identified in cr-osvp1 vs. NIP and cr-sdr4 vs. NIP groups, respectively, with a large number of overlapped DEGs between two groups. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of common DEGs in two groups showed that genes related to carbohydrate metabolic, nucleoside metabolic, amylase activity and plant hormone signal transduction were involved in the dormancy regulation. These results suggest that OsVP1 and Sdr4 play an important role in regulating seed dormancy by multiple metabolism and regulatory pathways. The systematic analysis of the transcriptional level changes provides theoretical basis for the research of seed dormancy and germination in rice.
Key Message We investigated the mechanism of the effect of different light qualities on the synthesis and regulation of mung bean sprouts. Light quality acts as a signal molecule, strongly enhancing polyphenol biosynthesis in sprouts. Abstract Mung bean (Vigna radiata) sprouts are a popular sprouting vegetable all over the world and are an excellent source of polyphenols with high antioxidant activity. This study investigated the effects of light qualities on the kinetic changes and metabolic regulation mechanism of light signal-mediating polyphenols in three mung bean sprout cultivars. Experimental results showed that three light qualities significantly enhanced the contents of caffeic acid, rutin, vitexin, genistin and delphinidin 3-glucoside. Interestingly, ferulic acid and vitexin responded selectively to blue light and red light, severally. Most genes involved in polyphenol biosynthesis were activated under different light quality conditions, resulting in an overaccumulation of phenylpropanoids. Pearson correlation analysis showed that PAL, F3H, F3’H and F3′5’H expression correlated highly with rutin, whereas ANS expression paralleled anthocyanin biosynthesis. Moreover, MYB111, MYB3, MYB4, MYB1 and MYC2 were critical regulators of polyphenol biosynthesis in mung bean sprouts. These changes were likely due to the changes in the expression of the photoreceptor genes CRY-D, PHOT2, PHYE and light response genes (PIF3 and HY5). Our results provide insights into polyphenol biosynthesis in sprouts and microgreens. Graphical Abstract
Key message The Arabidopsis Tubby-like protein (TLP) encoding gene, AtTLP2, plays intricate roles during ABA-dependent abiotic stress signalling, particularly salt and dehydration stress responses. Abstract TLPs (Tubby-like proteins) are a small group of eukaryotic proteins characterized by the presence of a Tubby domain. The plant TLPs have been widely shown to play important roles during abiotic stress signaling. In this study, we investigated the role of an Arabidopsis TLP, AtTLP2, in mediating abiotic stress responses. Both attlp2 null mutant and overexpression (OE) lines, in Arabidopsis, were studied which indicated the role of the gene also in development. The attlp2 mutant showed an overall dwarfism, while its overexpression caused enhanced growth. AtTLP2 localized to the plasma membrane (PM) and showed nuclear translocation in response to dehydration stress. The protein interacted with ASK1 and ASK2, but failed to show transactivation activity in yeast. AtTLP2 was transcriptionally induced by stress, caused by salt, dehydration and ABA. The attlp2 mutant was insensitive to ABA, but hypersensitive to oxidative stress at all stages of growth. ABA insensitivity conferred tolerance to salt and osmotic stresses at the germination and early seedling growth stages, but caused hypersensitivity to salt and drought stresses at advanced stages of growth. The OE lines were more sensitive to ABA, causing increased sensitivity to most stresses at the seed germination stage, but conferring tolerance to salt and osmotic stresses at more advanced stages of development. The stomata of the attlp2 mutant were less responsive to ABA and H2O2, while that of the OE lines exhibited greater sensitivity. Several ABA-regulated stress responsive marker genes were found to be downregulated in the mutant, but upregulated in the OE lines. The study establishes that AtTLP2 plays intricate roles in abiotic stress signaling, and the response may be largely ABA dependent.
Top-cited authors
Pramod Gupta
  • Trees for the Futures
Sumita Jha
  • University of Calcutta
Kazuo Nakashima
  • Japan International Research Center for Agricultural Sciences
Ingo Potrykus
  • ETH Zurich
Manoj Prasad
  • National Institute of Plant Genome Research