Comparative Physiological and Transcriptomic Analyses of Two Contrasting Pepper Genotypes under Salt Stress Reveal Complex Salt Tolerance Mechanisms in Seedlings.

As a glycophyte plant, pepper (Capsicum annuum L.) is widely cultivated worldwide, but its growth is susceptible to salinity damage, especially at the seedling stage. Here, we conducted a study to determine the physiological and transcriptional differences between two genotype seedlings (P300 and 323F3) with contrasting tolerance under salt stress. The P300 seedlings were more salt-tolerant and had higher K+ contents, higher antioxidase activities, higher compatible solutes, and lower Na+ contents in both their roots and their leaves than the 323F3 seedlings. During RNA-seq analysis of the roots, more up-regulated genes and fewer down-regulated genes were identified between salt-treated P300 seedlings and the controls than between salt-treated 323F3 and the controls. Many ROS-scavenging genes and several SOS pathway genes were significantly induced by salt stress and exhibited higher expressions in the salt-treated roots of the P300 seedlings than those of 323F3 seedlings. Moreover, biosynthesis of the unsaturated fatty acids pathway and protein processing in the endoplasmic reticulum pathway were deeply involved in the responses of P300 to salt stress, and most of the differentially expressed genes involved in the two pathways, including the genes that encode mega-6 fatty acid desaturases and heat-shock proteins, were up-regulated. We also found differences in the hormone synthesis and signaling pathway genes in both the P300 and 323F3 varieties under salt stress. Overall, our results provide valuable insights into the physiological and molecular mechanisms that affect the salt tolerance of pepper seedlings, and present some candidate genes for improving salt tolerance in pepper.

Melatonin Alleviates Copper Toxicity via Improving ROS Metabolism and Antioxidant Defense Response in Tomato Seedlings.

The excessive accumulation of copper (Cu2+) has become a threat to worldwide crop production. Recently, it was revealed that melatonin (MT) could play a crucial role against heavy metal (HM) stresses in plants. However, the underlying mechanism of MT function acted upon by Cu2+ stress (CS) has not been substantiated in tomatoes. In the present work, we produced MT-rich tomato plants by foliar usage of MT, and MT-deficient tomato plants by employing a virus-induced gene silencing methodology and exogenous foliar application of MT synthesis inhibitor para-chlorophenylalanine (pCPA). The obtained results indicate that exogenous MT meaningfully alleviated the dwarf phenotype and impeded the reduction in plant growth caused by excess Cu2+. Furthermore, MT effectively restricted the generation of reactive oxygen species (ROS) and habilitated cellular integrity by triggering antioxidant enzyme activities, especially via CAT and APX, but not SOD and POD. In addition, MT increased nonenzymatic antioxidant activity, including FRAP and the GSH/GSSG and ASA/DHA ratios. MT usage improved the expression of several defense genes (CAT, APX, GR and MDHAR) and MT biosynthesis-related genes (TDC, SNAT and COMT). Taken together, our results preliminarily reveal that MT alleviates Cu2+ toxicity via ROS scavenging, enhancing antioxidant capacity when subjected to excessive Cu2+. These results build a solid foundation for developing new insights to solve problems related to CS.

Silencing susceptibility genes in potato hinders primary infection of Phytophthora infestans at different stages.

Most potato cultivars are susceptible to late blight disease caused by the oomycete pathogen Phytophthora infestans. A new source of resistance to prevent or diminish pathogen infection is found in the genetic loss of host susceptibility. Previously, we showed that RNAi-mediated silencing of the potato susceptibility (S) genes StDND1, StDMR1 and StDMR6 leads to increased late blight resistance. The mechanisms underlying this S-gene mediated resistance have thus far not been identified. In this study, we examined the infection process of P. infestans on StDND1-, StDMR1- and StDMR6-silenced potato lines. Microscopic analysis showed that penetration of P. infestans spores was hampered on StDND1-silenced plants. On StDMR1- and StDMR6-silenced plants, P. infestans infection was arrested at a primary infection stage by enhanced cell death responses. Histochemical staining revealed that StDMR1- and StDMR6-silenced plants display elevated ROS levels in cells at the infection sites. Resistance in StDND1-silenced plants, however, seems not to rely on a cell death response as ROS accumulation was found to be absent at most inoculated sites. Quantitative analysis of marker gene expression suggests that the increased resistance observed in StDND1- and StDMR6-silenced plants relies on an early onset of SA- and ET-mediated signalling pathways. Resistance mediated by silencing StDMR1 was found to be correlated with the early induction of SA-mediated signalling. These data provide evidence that different defense mechanisms are involved in late blight resistance mediated by functional impairment of different potato S-genes.

Long-read sequencing and de novo assembly of the Luffa cylindrica (L.) Roem. genome.

Sponge gourd (Luffa cylindrica (L.) Roem.) or luffa is a diploid herbaceous plant with 26 chromosomes (2n = 26) and belongs to the family Cucurbitaceae. To address the limited knowledge of the genome of Luffa species, the chromosome-level genome of L. cylindrica was assembled and analysed using PacBio long reads and Hi-C data. We combined Hi-C data with a draft genome assembly to generate chromosome-length scaffolds. Thirteen scaffolds corresponding to the 13 chromosomes were assembled from 1,156 contigs to a final size of 669 Mb with a contig N50 size of 5 Mb and a scaffold N50 size of 53 Mb. After removing redundant sequences, 416.31 Mb (62.18% of the genome) of repeat sequences was detected. Subsequently, 31,661 protein-coding genes with an average of 5.69 exons per gene were identified in the L. cylindrica genome using de novo methods, transcriptome data and homologue-based approaches. In addition, 27,552 protein-coding genes (87.02%) were annotated in five databases. According to the phylogenetic analysis, L. cylindrica is closely related to Cucurbita and Cucumis species and diverged from their common ancestor ~28.6-67.1 million years ago. Genome collinearity analysis was performed in Cucurbita moschata, Cucumis sativus and L. cylindrica, and it demonstrated a high degree of conserved gene order in these three species. The completeness of the genome will provide high-quality genomic knowledge on breeding and reveal genetic variation in L. cylindrica.

Silencing of α-amylase StAmy23 in potato tuber leads to delayed sprouting.

Potato tuber dormancy is critical for the postharvest quality. The supply of carbohydrates is considered as one of the important factors controlling the rate of potato tuber sprouting. Starch is the major carbohydrate reserve in potato tuber, but very little is known about the specific starch degrading enzymes responsible for controlling tuber dormancy and sprouting. In this study, we demonstrate that an α-amylase gene StAmy23 is involved in starch breakdown and regulation of tuber dormancy. Silencing of StAmy23 delayed tuber sprouting by one to two weeks compared with the control. This phenotype is accompanied by reduced levels of reducing sugars and elevated levels of malto-oligosaccharides in tuber cortex and pith tissue below the bud eye of StAmy23-deficient potato tubers. Changes in soluble sugars is accompanied by a slight variation of phytoglycogen structure and starch granule size. Our results suggest that StAmy23 may stimulate sprouting by hydrolyzing soluble phytoglycogen to ensure supply of sugars during tuber dormancy.

The maize bHLH transcription factor bHLH105 confers manganese tolerance in transgenic tobacco.

Manganese (Mn) toxicity is an important limiting factor for crop production in acidic soils. The basic helix-loop-helix (bHLH) transcription factors are involved in a variety of physiological processes. However, whether the bHLHs are involved in excess Mn stress response is largely unknown. Here, we report the functional characterization of ZmbHLH105 isolated from maize (Zea mays). The transcript levels of ZmbHLH105 were higher in leaves, and were markedly up-regulated under excess Mn stress in maize. ZmbHLH105 was localized in the nucleus with transactivation activity. Ectopic expression of ZmbHLH105 enhanced Mn tolerance in Saccharomyces cerevisiae cells. ZmbHLH105-overexpressing (OE) plants showed improved excess Mn tolerance in transgenic tobacco. The stress-tolerant phenotypes of these OE tobacco lines were accompanied by increases of key antioxidant enzyme activities, but decreases of reactive oxygen species (ROS) accumulations. Importantly, the OE plants had less increases than the wild-type in toxic Mn accumulation. Moreover, the transcript levels of Mn/Fe-related transporters in the OE lines displayed remarkable decreases compared with the wild-type under Mn stress, suggesting that ZmbHLH105 reduced Mn accumulation in plants largely by repressing expression of Mn/Fe-regulated transporter genes. Taken together, these results indicate that ZmbHLH105 confers improved Mn stress tolerance possibly by regulating antioxidant machinery-mediated ROS scavenging and expression of Mn/Fe-related transporters in plants. ZmbHLH105 could be exploited for developing drought-tolerant maize varieties.

Silencing of DND1 in potato and tomato impedes conidial germination, attachment and hyphal growth of Botrytis cinerea.

BACKGROUND: Botrytis cinerea, a necrotrophic pathogenic fungus, attacks many crops including potato and tomato. Major genes for complete resistance to B. cinerea are not known in plants, but a few quantitative trait loci have been described in tomato. Loss of function of particular susceptibility (S) genes appears to provide a new source of resistance to B. cinerea in Arabidopsis. RESULTS: In this study, orthologs of Arabidopsis S genes (DND1, DMR6, DMR1 and PMR4) were silenced by RNAi in potato and tomato (only for DND1). DND1 well-silenced potato and tomato plants showed significantly reduced diameters of B. cinerea lesions as compared to control plants, at all-time points analysed. Reduced lesion diameter was also observed on leaves of DMR6 silenced potato plants but only at 3 days post inoculation (dpi). The DMR1 and PMR4 silenced potato transformants were as susceptible as the control cv Desiree. Microscopic analysis was performed to observe B. cinerea infection progress in DND1 well-silenced potato and tomato leaves. A significantly lower number of B. cinerea conidia remained attached to the leaf surface of DND1 well-silenced potato and tomato plants and the hyphal growth of germlings was hampered. CONCLUSIONS: This is the first report of a cytological investigation of Botrytis development on DND1-silenced crop plants. Silencing of DND1 led to reduced susceptibility to Botrytis, which was associated with impediment of conidial germination and attachment as well as hyphal growth. Our results provide new insights regarding the use of S genes in resistance breeding.

Silencing of six susceptibility genes results in potato late blight resistance.

Phytophthora infestans, the causal agent of late blight, is a major threat to commercial potato production worldwide. Significant costs are required for crop protection to secure yield. Many dominant genes for resistance (R-genes) to potato late blight have been identified, and some of these R-genes have been applied in potato breeding. However, the P. infestans population rapidly accumulates new virulent strains that render R-genes ineffective. Here we introduce a new class of resistance which is based on the loss-of-function of a susceptibility gene (S-gene) encoding a product exploited by pathogens during infection and colonization. Impaired S-genes primarily result in recessive resistance traits in contrast to recognition-based resistance that is governed by dominant R-genes. In Arabidopsis thaliana, many S-genes have been detected in screens of mutant populations. In the present study, we selected 11 A. thaliana S-genes and silenced orthologous genes in the potato cultivar Desiree, which is highly susceptible to late blight. The silencing of five genes resulted in complete resistance to the P. infestans isolate Pic99189, and the silencing of a sixth S-gene resulted in reduced susceptibility. The application of S-genes to potato breeding for resistance to late blight is further discussed.

Down-regulation of Arabidopsis DND1 orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew.

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.

The maize AAA-type protein SKD1 confers enhanced salt and drought stress tolerance in transgenic tobacco by interacting with Lyst-interacting protein 5.

ATPase associated with various cellular activities (AAA) proteins are important regulators involved in diverse cellular functions. To date, the molecular mechanisms of AAA proteins involved in response to salt and drought stresses in plants are largely unknown. In this study, a putative SKD1 (suppressor of K(+) transport growth defect 1) ortholog from Zea mays (ZmSKD1), which encodes a putative AAA protein, was isolated. The transcript levels of ZmSKD1 were higher in aerial tissues and were markedly up-regulated by salt or drought stress. Over-expression of ZmSKD1 in tobacco plants enhanced their tolerances not only to salt but to drought. Moreover, reactive oxygen species accumulations in ZmSKD1 transgenic lines were relative less than those in wild-type plants during salt or PEG-induced water stress. The interaction between ZmSKD1 and NtLIP5 (Lyst-Interacting Protein 5 homolog from Nicotiana tabacum) was confirmed by both yeast two-hybrid and immuno-precipitation assays; moreover, the α-helix-rich domain in the C-terminus of ZmSKD1 was identified to be required for its interaction with NtLIP5 using truncation mutations. Collectively, these data demonstrate that ZmSKD1could be involved in salt and drought stress responses and its over-expression enhances salt or drought stress tolerance possibly through interacting with LIP5 in tobacco. This study may facilitate our understandings of the biological roles of SKD1-mediated ESCRT pathway under stress conditions in higher plants and accelerate genetic improvement of crop plants tolerant to environmental stresses.

Overexpression of a maize sulfite oxidase gene in tobacco enhances tolerance to sulfite stress via sulfite oxidation and CAT-mediated H2O2 scavenging.

Sulfite oxidase (SO) plays an important role in sulfite metabolism. To date, the molecular mechanisms of sulfite metabolism in plants are largely unknown. Previously, a full-length cDNA of the putative sulfite oxidase gene from maize (ZmSO) was cloned, and its response to SO(2)/sulfite stress at the transcriptional level was characterized. In this study, the recombinant ZmSO protein was purified from E. coli. It exhibited sulfite-dependent activity and had strong affinity for the substrate sulfite. Over-expression (OE) of ZmSO in tobacco plants enhanced their tolerance to sulfite stress. The plants showed much less damage, less sulfite accumulation, but greater amounts of sulfate. This suggests that tolerance of transgenic plants to sulfite was enhanced by increasing SO expression levels. Interestingly, H(2)O(2) accumulation levels by histochemical detection and quantitative determination in the OE plants were much less than those in the wild-type upon sulfite stress. Furthermore, reductions of catalase levels detected in the OE lines were considerably less than in the wild-type plants. This indicates that SO may play an important role in protecting CAT from inhibition by excess sulfite. Collectively, these data demonstrate that transgenic tobacco plants over-expressing ZmSO enhance tolerance to excess sulfite through sulfite oxidation and catalase-mediated hydrogen peroxide scavenging. This is the first SO gene from monocots to be functionally characterized.

The movement protein of barley yellow dwarf virus-GAV self-interacts and forms homodimers in vitro and in vivo.

The 17-kDa movement protein (MP) of the GAV strain of barley yellow dwarf virus (BYDV-GAV) can bind the viral RNA and target to the nucleus. However, much less is known about the active form of the MP in planta. In this study, the ability of the MP to self-interact was analyzed by yeast two-hybrid assay and bimolecular fluorescence complementation. The BYDV-GAV MP has a strong potential to self-interact in vitro and in vivo, and self-interaction was mediated by the N-terminal domain spanning the second α-helix (residues 17-39). Chemical cross-linking and heterologous MP expression from a pea early browning virus (PEBV) vector further showed that MP self-interacts to form homodimers in vitro and in planta. Interestingly, the N-terminal domain necessary for MP self-interaction has previously been identified as important for nuclear targeting. Based on these findings, a functional link between MP self-interaction and nuclear targeting is discussed.