Transient Expression Analysis Research Articles

Ancient duplications, multidimensional specializations, and defense role of hexokinases in wheat.

Hexokinases (HXKs), which sense and catalyze cellular sugar, play a critical role in the growth and development of various plants, including wheat, a primary source of human calories frequently attacked by fungal pathogens. However, the evolutionary dynamics and functional diversification of HXKs in wheat, particularly their roles in plant defense, remain unclear. Here, we discovered that the wheat hexokinase gene family originated through multiple ancient gene duplications across different plant lineages and has undergone comprehensive, multidimensional functional specialization in gene expression, subcellular localization, enzyme activity, and regulation of plant defense responses. Gene expression analysis suggests that two-thirds of the TaHXK genes are responsive to fungal infection. Subcellular analysis reveals that while six TaHXKs are localized in mitochondria, three TaHXKs from different phylogenetic branches are sorted into other cellular compartments. Notably, biochemical analysis shows that TaHXKs in mitochondria differ in their glucose-catalyzing activity, with TaHXK5 and TaHXK3 exhibiting the highest and lowest enzyme activity, respectively. Consistently, transient expression analysis suggests that TaHXK5 induces various plant defense responses, while TaHXK3 is defective in activating some plant defense responses. Furthermore, inactivation of the glucokinase activity of TaHXK5 compromised its function in defense activation, suggesting that mitochondrial TaHXKs display functional divergence in both enzyme activity and defense-inducing activity that are intrinsically connected. Overall, our findings reveal that the multidimensional specialization events following the ancient duplication events may have shaped the functional diversity of HXKs in wheat, shedding light on their evolutionary dynamics and potentially contributing to the improvement of wheat defense.

Rehmannia glutinosa RgMATE35 Participates in the Root Secretion of Phenolic Acids and Modulates the Development of Plant Replant Disease.

Phenolic allelochemicals from root exudates dominate rhizosphere formation, lead to autotoxicity in plants subjected to continuous monoculture (CM) stress and induce the emergence of replant disease. However, the regulatory mechanisms governing the transport of phenolics from plant roots to the rhizosphere remain poorly understood. A potential phenolic efflux transporter from Rehmannia glutinosa, designated RgMATE35, has been preliminarily characterized. The objective of this study was to elucidate the molecular function of RgMATE35 in the secretion of phenolics and to investigate its role in the development of plant replant disease using quantitative real-time PCR (qRT-PCR), genetic transformation, HPLC-Q-TOF-MS and other analytical techniques. A tissue expression pattern analysis of RgMATE35 revealed that it is highly expressed in plant roots. Transient expression analysis confirmed the localization of the protein in plasma membranes. An assessment of the transport activity of RgMATE35 in Xenopus oocytes indicated that it plays a role in facilitating the efflux of labeled ferulic acid ([2H3]-FA) and trans-p-coumaric acid [2H6]-pCA. The results of functional studies in R. glutinosa demonstrated that RgMATE35 positively mediates the secretion of FA and pCA from plant roots into the rhizosphere. A molecular and physiological analysis of RgMATE35 transgenic plants subjected to CM stress revealed that the overexpression or repression of RgMATE35 resulted in notable changes in the degree of autotoxic injury in plants. These findings demonstrate that RgMATE35 plays a positive role in the development of replant disease through the secretion of phenolic acids from plant roots. They also provide a fundamental framework for elucidating the molecular regulatory mechanism through which MATEs regulate replant disease through the root secretion of allelochemicals.

Multi-omics analysis reveals anthocyanin synthesis is associated with drought stress tolerance in Chaenomeles speciosa flowers

Drought is one of the most severe abiotic stresses that limit plant growth. Chaenomeles speciosa, a Rosaceae plant of high ornamental value, exhibits noticeable changes in petal color under drought conditions. A multi-omics joint analysis was employed to examine the mechanism of flower color changes under drought conditions. We detected that the main reason for the change in flower color is the synthesis of anthocyanins, while ethylene (ET) and abscisic acid (ABA) also significantly increase. While ET and ABA are known to promote anthocyanin synthesis, their coordinated regulation under drought conditions remains unclear. This study aims to validate the hypothesis that ET/ABA mediated responses to drought regulate anthocyanin synthesis. The study inferred that the CsERF2, CsbZIP24–CsMYB6 signaling module plays a crucial role in the precise and dynamic regulation of anthocyanin synthesis. Yeast heterologous expression is used to demonstrate the drought tolerance function of candidate genes. Also, we performed transient expression analysis in petals suggests that these genes significantly contribute to anthocyanin accumulation under drought conditions. Moreover, Yeast one-hybrid and dual luciferase assays indicated that CsERF2 and CsbZIP24 can directly activate the expression of CsMYB6. Yeast two-hybrid, bimolecular fluorescence complementation, and Split-luciferase assays revealed interactions between CsERF2 and CsbZIP24 proteins, promoting anthocyanin biosynthesis. The study proposes a regulatory model for the mechanism of flower color change and aimed at exploring drought resistant ornamental plants and achieving their sustainable development.

Pathophysiological significance of the p.E31G variant in RAC1 responsible for a neurodevelopmental disorder with microcephaly

RAC1 encodes a Rho family small GTPase that regulates actin cytoskeletal reorganization and intracellular signaling pathways. Pathogenic RAC1 variants lead to a neurodevelopmental disorder with diverse phenotypic manifestations, including abnormalities in brain size and facial dysmorphism. However, the underlying pathophysiological mechanisms have yet to be elucidated. Here, we present the case of a school-aged male who exhibited global developmental delay, intellectual disability, and acquired microcephaly. Through whole exome sequencing, we identified a novel de novo variant in RAC1, (NM_006908.5): c.92 A > G,p.(E31G). We then examined the pathophysiological significance of the p.E31G variant by focusing on brain development. Biochemical analyses revealed that the recombinant RAC1-E31G had no discernible impact on the intrinsic GDP/GTP exchange activity. However, it exhibited a slight inhibitory effect on GTP hydrolysis. Conversely, it demonstrated a typical response to both a guanine-nucleotide exchange factor and a GTPase-activating protein. In transient expression analyses using COS7 cells, RAC1-E31G exhibited minimal interaction with the downstream effector PAK1, even in its GTP-bound state. Additionally, overexpression of RAC1-E31G was observed to exert a weak inhibitory effect on the differentiation of primary cultured hippocampal neurons. Moreover, in vivo studies employing in utero electroporation revealed that acute expression of RAC1-E31G resulted in impairments in axonal elongation and dendritic arborization in the young adult stage. These findings suggest that the p.E31G variant functions as a dominant-negative version in the PAK1-mediated signaling pathway and is responsible for the clinical features observed in the patient under investigation, namely microcephaly and intellectual disability.

The effect of nitrosative stress on histone H3 and H4 acetylation in Phytophthora infestans life cycle

The oomycete Phytophthora infestans is one of the most destructive phytopathogens globally. It has a proven ability to adapt to changing environments rapidly; however, molecular mechanisms responsible for host invasion and adaptation to new environmental conditions still need to be explored. The study aims to understand the epigenetic mechanisms exploited by P. infestans in response to nitrosative stress conditions created by the (micro)environment and the host plant. To characterize reactive nitrogen species (RNS)-dependent acetylation profiles in avirulent/virulent (avr/vr) P. infestans, a transient gene expression, ChIP and immunoblot analyses, and nitric oxide (NO) emission by chemiluminescence were used in combination with the pharmacological approach. Nitrosative stress increased total H3/H4 acetylation and some histone acetylation marks, mainly in sporulating hyphae of diverse (avr/vr) isolates and during potato colonization. These results correlated with transcriptional up-regulation of acetyltransferases PifHAC3 and PifHAM1, catalyzing H3K56 and H4K16 acetylation, respectively. NO or peroxynitrite–mediated changes were also associated with H3K56 and H4K16 mark deposition on the critical pathogenicity-related gene promoters (CesA1, CesA2, CesA3, sPLD-like1, Hmp1, and Avr3a) elevating their expression. Our study highlights RNS-dependent transcriptional reprogramming via histone acetylation of essential gene expression in the sporulating and biotrophic phases of plant colonization by P. infestans as a tool promoting its evolutionary plasticity.

Spatial organization of putrescine synthesis in plants

Three plant pathways for the synthesis of putrescine have been described to date. These are the synthesis of putrescine from ornithine, by ornithine decarboxylase (ODC); the synthesis of putrescine from arginine by arginine decarboxylase, agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (NLP1); and arginine decarboxylase and agmatinase. To address how these pathways are organized in plants, we have used transient expression analysis of these genes in the leaves of Nicotiana benthamiana. Brassicas do not have ODC, but the single ODC gene from rice and one of the soybean genes, were localized to the ER. Transient expression of the rice agmatinase gene showed that it was localized to the mitochondria. In A. thaliana there are five isoforms of AIH and three isoforms of NLP1. Stable GFP-tagged transformants of the longest isoforms of AIH and NLP1 showed that both proteins were localized to the ER, but in tissues with chloroplasts, the localization was concentrated to lamellae adjacent to chloroplasts. Transient expression analyses showed that four of the isoforms of AIH and all of the isoforms of NLP1 were localized to the ER. However, AIH.4 was localized to the chloroplast. Combining these results with other published data, reveal that putrescine synthesis is excluded from the cytoplasm and is spatially localized to the chloroplast, ER, and likely the mitochondria. Synthesis of putrescine in the ER may facilitate cell to cell transport via plasmodesmata, or secretion via vesicles. Differential expression of these pathways may enable putrescine-mediated activation of hormone-responsive genes.

DEK219 and HSF17 Collaboratively Regulate the Kernel Length in Maize

The kernel length is a crucial determinant of maize (Zea mays L.) yield; however, only a limited number of genes regulating kernel length have been validated, thus leaving our understanding of the mechanisms governing kernel length incomplete. We previously identified a maize kernel mutant, defective kernel219 (dek219), which encodes the DICER-LIKE1 protein that is essential for miRNA biogenesis. The present study revealed that dek219 consistently exhibits a stable phenotype characterized by a reduced kernel length. Further analysis indicated that dek219 may reduce the kernel length by inhibiting the expression of genes involved in regulating kernel length. By employing miRNA-target gene prediction, expression analysis, and correlation analysis, we successfully identified nine transcription factors that potentially participate in the regulation of kernel length under the control of DEK219. Among them, the upregulation fold change of HEAT SHOCK TRANSCRIPTION FACTOR17 (HSF17) expression was the highest, and the difference was most significant. The results of transient expression analysis and electrophoretic mobility shift assay (EMSA) indicated that HSF17 can inhibit the expression of DEFECTIVE ENDOSPERM18 (DE18), a gene involved in regulating kernel length. Furthermore, the hsf17 mutant exhibited a significant increase in kernel length, suggesting that HSF17 functions as a negative regulator of kernel length. The results of this study provide crucial evidence for further elucidating the molecular regulatory mechanism underlying maize kernel length and also offer valuable genetic resources for breeding high-yielding maize varieties.

Screening and functional verification of drought resistance-related genes in castor bean seeds

Drought is one of the natural stresses that greatly impact plants. Castor bean (Ricinus communis L.) is an oil crop with high economic value. Drought is one of the factors limiting castor bean growth. The drought resistance mechanisms of castor bean have become a research focus. In this study, we used castor germinating embryos as experimental materials, and screened genes related to drought resistance through physiological measurements, proteomics and metabolomics joint analysis; castor drought-related genes were subjected to transient silencing expression analysis in castor leaves to validate their drought-resistant functions, and heterologous overexpression and backward complementary expression in Arabidopsis thaliana, and analysed the mechanism of the genes' response to the participation of Arabidopsis thaliana in drought-resistance.Three drought tolerance-related genes, RcECP 63, RcDDX 31 and RcA/HD1, were obtained by screening and analysis, and transient silencing of expression in castor leaves further verified that these three genes corresponded to drought stress, and heterologous overexpression and back-complementary expression of the three genes in Arabidopsis thaliana revealed that the function of these three genes in drought stress response.In this study, three drought tolerance related genes, RcECP 63, RcDDX 31 and RcA/HD1, were screened and analysed for gene function, which were found to be responsive to drought stress and to function in drought stress, laying the foundation for the study of drought tolerance mechanism in castor bean.

The transcriptional regulation of a putative hemicellulose gene, PtrPARVUS2 in poplar

The plant cell wall serves as a critical interface between the plant and its environment, offering protection against various stresses and contributing to biomass production. Hemicellulose is one of the major components of the cell wall, and understanding the transcriptional regulation of its production is essential to fully understanding cell wall formation. This study explores the regulatory mechanisms underlying one of the genes involved in hemicellulose biosynthesis, PtrPARVUS2. Six transcription factors (TFs) were identified from a xylem-biased library to negatively regulate PtrPARVUS2 expression. These TFs, belonging to diverse TF families, were confirmed to bind to specific cis-elements in the PtrPARVUS2 promoter region, as validated by Yeast One-Hybrid (Y1H) assays, transient expression analysis, and Chromatin Immunoprecipitation sequencing (ChIP-seq) assays. Furthermore, motif analysis identified putative cis-regulatory elements bound by these TFs, shedding light on the transcriptional regulation of SCW biosynthesis genes. Notably, several TFs targeted genes encoding uridine diphosphate glycosyltransferases (UGTs), crucial enzymes involved in hemicellulose glycosylation. Phylogenetic analysis of UGTs regulated by these TFs highlighted their diverse roles in modulating hemicellulose synthesis. Overall, this study identifies a set of TFs that regulate PARVUS2 in poplar, providing insights into the intricate coordination of TFs and PtrPARVUS2 in SCW formation. Understanding these regulatory mechanisms enhances our ability to engineer plant biomass for tailored applications, including biofuel production and bioproduct development.

Insights into the PYR/PYL/RCAR Gene Family in Pomegranates (Punica granatum L.): A Genome-Wide Study on Identification, Evolution, and Expression Analysis

The response of plants to abiotic stress is intricately mediated by PYR/PYL/RCARs, key components within the ABA signal transduction pathway. Despite the widespread identification of PYL genes across diverse plant species, the evolutionary history and structural characteristics of these genes within the pomegranate (Punica granatum L.) remained unexplored. In this study, we uncovered, for the first time, 12 PgPYLs from the whole genome dataset of ‘Tunisia’, mapping them onto five chromosomes and categorizing them into three distinct subgroups (Group I, Group II, and Group III) through phylogenetic analysis. Detailed examination of the composition of these genes revealed similar conserved motifs and exon–intron structures among genes within the same subgroup. Fragment duplication emerged as the primary mechanism driving the amplification of the PYL gene family, as evidenced by intra-species collinearity analysis. Furthermore, inter-species collinearity analysis provided insights into potential evolutionary relationships among the identified PgPYL genes. Cis-acting element analysis revealed a rich repertoire of stress and hormone response elements within the promoter region of PgPYLs, emphasizing their putative roles in diverse signaling pathways. Upon treatment with 100 μmol/L ABA, we investigated the expression patterns of the PgPYL gene family, and the qRT-PCR data indicated a significant up-regulation in the majority of PYL genes. This suggested an active involvement of PgPYL genes in the plant’s response to exogenous ABA. Among them, PgPYL1 was chosen as a candidate gene to explore the function of the gene family, and the CDS sequence of PgPYL1 was cloned from pomegranate leaves with a full length of 657 bp, encoding 218 amino acids. Tobacco transient expression analysis demonstrated a consistent trend in the expression levels of pBI121-PgPYL1 and the related genes of the ABA signaling pathway, both of which increased initially before declining. This study not only contributes to the elucidation of the genomic and structural attributes of PgPYL genes but also provides a foundation for understanding their potential functions in stress responses. The identified conserved motifs, evolutionary relationships, and expression patterns under ABA treatment pave the way for further research into the PgPYL gene family’s role in pomegranate biology, offering valuable insights for future studies on genetic improvement and stress resilience in pomegranate cultivation.

Transcriptome analysis reveals various genes involved in the regulation of potato to late blight

BackgroundPotato (Solanum tuberosum L.) production is seriously threatened by the oomycete Phytophthora infestans (P. infestans). However, it remains unclear how the two potato cultivars, Q9 (moderately resistant) and Atl (susceptible), initiate distinct defense responses after inoculation with P. infestans. To uncover the underlying process, we investigated the dynamic gene expression profiles and the functions of the differentially expressed genes (DEGs).ResultsThe results showed that the numbers of up-regulated DEGs were 1345, 1063 and 1694 in Q9 and 272, 603 and 2554 in Atl at 1 day post inoculation (dpi), 3 dpi and 5 dpi, respectively. Meanwhile, the down-regulated DEGs were 1557, 1417 and 389 in Q9 and 114, 364 and 2528 in Atl simultaneously. KEGG enrichment analysis revealed that plant-pathogen interaction, phenylpropanoid biosynthesis and MAPK signaling pathway were significantly enriched in Q9 at the later stage. Furthermore, DEGs associated with plant-pathogen interaction and phenylpropanoid biosynthesis showed higher expression levels in Q9 compared to Atl, especially at the later stage. The expressions of eight DEGs were validated by quantitative real-time PCR (qRT-PCR), which further verified the accuracy of the transcriptomics analysis. In addition, DEGs that were exclusively up-regulated and down-regulated in Q9 were analyzed, which may contribute to the resistance of Q9. Transient expression analysis revealed that four DEGs, including StHP1, StMYB2, StHSP3 and StNAC5, exhibited increased tolerance to P. infestans, indicating a positive role in enhancing the resistance of Q9.ConclusionTherefore, the DEGs associated with plant-pathogen interaction, phenylpropanoid biosynthesis and MAPK signaling pathway were involved in regulating late blight, and the expression level of the related genes significantly increased in Q9. In addition, StHP1, StHSP3 and the transcription factors, including StMYB2 and StNAC5, played positive roles in improving the resistance of potato Q9 to P. infestans. These findings provide useful information for further understanding the molecular mechanism of potato resistance to late blight. Furthermore, our study identified novel resistance genes that can be used in resistance breeding and functional research.Graphical abstract

TaCRT3 Is a Positive Regulator of Resistance to Blumeria graminis f. sp. tritici in Wheat.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most prevalent diseases of wheat worldwide and can lead to severe yield reductions. Identifying genes involved in powdery mildew resistance will be useful for disease resistance breeding and control. Calreticulin (CRT) is a member of multigene family widely found in higher plants and is associated with a variety of plant physiological functions and defense responses. However, the role of CRT in wheat resistance to powdery mildew remains unclear. TaCRT3 was identified from the proteomic sequence of an incompatible interaction between the wheat (Triticum aestivum) cultivar Xingmin 318 and the Bgt isolate E09. Following analysis of transient expression of the GFP-TaCRT3 fusion protein in Nicotiana benthamiana leaves, TaCRT3 was localized in the nucleus, cytoplasm, and cell membrane. Transcript expression levels of TaCRT3 were significantly upregulated in the wheat-Bgt incompatible interaction. More critically, knockdown of TaCRT3 using virus-induced gene silencing resulted in attenuated resistance to Bgt in wheat. Histological analysis showed a significant increase in Bgt development in TaCRT3-silenced plants, whereas the pathogen-related gene was significantly downregulated in TaCRT3-silenced leaves. In addition, overexpression of TaCRT3 in wheat enhanced the resistance to powdery mildew, the growth of Bgt was significantly inhibited, and the area of H2O2 near the infection site and the expression of defense-related genes of the salicylic acid pathway significantly increased. These findings imply that TaCRT3 may act as a disease resistance factor that positively regulates resistance to powdery mildew, during which SA signaling is probably activated.

Genome-wide characterization and function analysis of ginger (Zingiber officinale Roscoe) ZoGRFs in responding to adverse stresses

Growth-regulating factors (GRFs) play crucial roles in plant growth, development, hormone signaling, and stress response. Despite their significance, the roles of GRFs in ginger remain largely unknown. Herein, 31 ginger ZoGRFs were identified and designated as ZoGRF1–ZoGRF31 according to their phylogenetic relationships. All ZoGRFs were characterized as unstable, hydrophilic proteins, with 29 predicted to be located in the nucleus. Functional cis-elements related to growth and development were enriched in ZoGRF's promoter regions. RNA-seq and RT-qPCR analysis revealed that ZoGRF12, ZoGRF24, and ZoGRF28 were highly induced in various growth and development stages, displaying differential regulation under waterlogging, chilling, drought, and salt stresses, indicating diverse expression patterns of ZoGRFs. Transient expression analysis in Nicotiana benthamiana indicated that overexpressing ZoGRF28 regulated the transcription levels of salicylic acid, jasmonic acid, and pattern-triggered immunity-related genes, increased chlorophyll content and contributed to reduced disease lesions and an increased net photosynthetic rate. This research lays the foundation for further understanding the biological roles of ZoGRFs.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

Nuclear Localization of G3BP6 Is Essential for the Flowering Transition in Arabidopsis.

The Ras GTPase-activating protein SH3 domain-binding protein (G3BP) belongs to the highly conserved family of RNA-binding proteins, which has been well-investigated in humans and animals. However, limited study of plant G3BP has been reported, and the precise biological function of the G3BP family has not been elucidated yet. In this study, the Arabidopsis G3BP family, comprising seven members, was comparatively analyzed. Transcriptome analysis showed that most G3BP genes are ubiquitously expressed in various tissues/organs. Transient expression analysis revealed that all G3BPs were presented in the cytoplasm, among which G3BP6 was additionally found in the nucleus. Further study revealed a conserved NLS motif required for the nuclear localization of G3BP6. Additionally, phenotypic analysis revealed that loss-of-function g3bp6 presented late-flowering phenotypes. RNA-sequencing analysis and qRT-PCR assays demonstrated that the expressions of abundant floral genes were significantly altered in g3bp6 plants. We also discovered that overexpression of G3BP6 in the nucleus, rather than in the cytoplasm, propelled bolting. Furthermore, we revealed that the scaffold protein Receptor for Activated C Kinase 1 (RACK1) interacted with and modulated the nuclear localization of G3BP6. Altogether, this study sheds new light on G3BP6 and its specific role in regulating the flowering transition in Arabidopsis.

HaMYBA-HabHLH1 regulatory complex and HaMYBF fine-tune red flower coloration in the corolla of sunflower (Helianthus annuus L.)

Sunflowers are well-known ornamental plants, while sunflowers with red corolla are rare and the mechanisms underlying red coloration remain unclear. Here, a comprehensive analysis of metabolomics and transcriptomics on flavonoid pathway was performed to investigate the molecular mechanisms underlying the differential color formation between red sunflower Pc103 and two yellow sunflowers (Yr17 and Y35). Targeted metabolomic analysis revealed higher anthocyanin levels but lower flavonol content in Pc103 compared to the yellow cultivars. RNA-sequencing and phylogenetic analysis identified multiple genes involved in the flavonoid pathway, including series of structural genes and three MYB and bHLH genes. Specifically, HaMYBA and HabHLH1 were up-regulated in Pc103, whereas HaMYBF exhibited reduced expression. HaMYBA was found to interact with HabHLH1 in vivo and in vitro, while HaMYBF does not. Transient expression analysis further revealed that HabHLH1 and HaMYBA cooperatively regulate increased expression of dihydroflavonol 4-reductase (DFR), leading to anthocyanin accumulation. On the other hand, ectopic expression of HaMYBF independently modulates flavonol synthase (FLS) expression, but hindered anthocyanin production. Collectively, our findings suggest that the up-regulation of HaMYBA and HabHLH1, as well as the down-regulation of HaMYBF, contribute to the red coloration in Pc103. It offers a theoretical basis for improving sunflower color through genetic engineering.

MeGLYI-13, a Glyoxalase I Gene in Cassava, Enhances the Tolerance of Yeast and Arabidopsis to Zinc and Copper Stresses.

Although zinc and copper are the two essential nutrients necessary for plant growth, their excessive accumulation in soil not only causes environmental pollution but also seriously threatens human health and inhibits plant growth. The breeding of plants with novel zinc or copper toxicity tolerance capacities represents one strategy to address this problem. Glyoxalase I (GLYI) family genes have previously been suggested to be involved in the resistance to a wide range of abiotic stresses, including those invoked by heavy metals. Here, a MeGLYI-13 gene cloned from a cassava SC8 cultivar was characterized with regard to its potential ability in resistance to zinc or copper stresses. Sequence alignment indicated that MeGLYI-13 exhibits sequence differences between genotypes. Transient expression analysis revealed the nuclear localization of MeGLYI-13. A nuclear localization signal (NLS) was found in its C-terminal region. There are 12 Zn2+ binding sites and 14 Cu2+ binding sites predicted by the MIB tool, of which six binding sites were shared by Zn2+ and Cu2+. The overexpression of MeGLYI-13 enhanced both the zinc and copper toxicity tolerances of transformed yeast cells and Arabidopsis seedlings. Taken together, our study shows the ability of the MeGLYI-13 gene to resist zinc and copper toxicity, which provides genetic resources for the future breeding of plants resistant to zinc and copper and potentially other heavy metals.

Hydroxypyruvate reductase gene family in Nicotiana benthamiana: Genome-wide identification and expression pattern profiling

Hydroxypyruvate (HP), the key intermediate of photorespiration, is converted to glycerate via the catalysis of hydroxypyruvate reductases (HPRs) with NADH/NADPH as cofactors. The non-lethal phenotype resulting from HPR defects allows for the use of mutants to investigate interactions between photorespiration and other cellular processes, facilitating the establishment of plant chassis with compromised photorespiration. Considering that establishing a plant chassis of the HPR- series would provide great potential in promoting plant synthetic biology to tackle future challenges, the genome-wide identification and bioinformatics analysis of HPR gene family in N. benthamiana are imperative but remains to be solved. In this study, 12 N. benthamiana hydroxypyruvate reductases (NbHPRs) were identified from a genome-wide study. These genes could be classified into three subclasses by phylogenetic analysis, and conserved gene structures or motif compositions were identified in each subclass. A variety of signal-sensing elements were identified in the HPRs promoter regions indicating their regulation by multiple potential transcription factors such as C2H2 proteins. Quantitative real-time PCR (qRTPCR) results further demonstrated the higher expression levels of NbHPRs in functional and young leaves compared to other organs. Subsequently, we confirmed the subcellular localization of NbHPRs with transient expression analysis, which suggests their different functions. Moreover, the relative expression level of the gene under nitrogen (N) treatment was assessed through qRTPCR analysis. These works will offer valuable insights into elucidating the function and mechanism of HPRs in N. benthamiana, thus illuminating the strategies for introducing artificial carbon fixation pathways to tackle future challenges with the modification on photorespiration.

A fungal CFEM-containing effector targets NPR1 regulator NIMIN2 to suppress plant immunity.

Colletotrichum fructicola causes a broad range of plant diseases worldwide and secretes many candidate proteinous effectors during infection, but it remains largely unknown regarding their effects in conquering plant immunity. Here, we characterized a novel effector CfEC12 that is required for the virulence of C. fructicola. CfEC12 contains a CFEM domain and is highly expressed during the early stage of host infection. Overexpression of CfEC12 suppressed BAX-triggered cell death, callose deposition and ROS burst in Nicotiana benthamiana. CfEC12 interacted with apple MdNIMIN2, a NIM1-interacting (NIMIN) protein that putatively modulates NPR1 activity in response to SA signal. Transient expression and transgenic analyses showed that MdNIMIN2 was required for apple resistance to C. fructicola infection and rescued the defence reduction in NbNIMIN2-silenced N. benthamiana, supporting a positive role in plant immunity. CfEC12 and MdNPR1 interacted with a common region of MdNIMIN2, indicating that CfEC12 suppresses the interaction between MdNIMIN2 and MdNPR1 by competitive target binding. In sum, we identified a fungal effector that targets the plant salicylic acid defence pathway to promote fungal infection.

Genome-Wide Identification of GmSPS Gene Family in Soybean and Expression Analysis in Response to Cold Stress.

Sucrose metabolism plays a critical role in development, stress response, and yield formation of plants. Sucrose phosphate synthase (SPS) is the key rate-limiting enzyme in the sucrose synthesis pathway. To date, genome-wide survey and comprehensive analysis of the SPS gene family in soybean (Glycine max) have yet to be performed. In this study, seven genes encoding SPS were identified in soybean genome. The structural characteristics, phylogenetics, tissue expression patterns, and cold stress response of these GmSPSs were investigated. A comparative phylogenetic analysis of SPS proteins in soybean, Medicago truncatula, Medicago sativa, Lotus japonicus, Arabidopsis, and rice revealed four families. GmSPSs were clustered into three families from A to C, and have undergone five segmental duplication events under purifying selection. All GmSPS genes had various expression patterns in different tissues, and family A members GmSPS13/17 were highly expressed in nodules. Remarkably, all GmSPS promoters contain multiple low-temperature-responsive elements such as potential binding sites of inducer of CBF expression 1 (ICE1), the central regulator in cold response. qRT-PCR proved that these GmSPS genes, especially GmSPS8/18, were induced by cold treatment in soybean leaves, and the expression pattern of GmICE1 under cold treatment was similar to that of GmSPS8/18. Further transient expression analysis in Nicotiana benthamiana and electrophoretic mobility shift assay (EMSA) indicated that GmSPS8 and GmSPS18 transcriptions were directly activated by GmICE1. Taken together, our findings may aid in future efforts to clarify the potential roles of GmSPS genes in response to cold stress in soybean.