Identification and functional characterization of conserved cis-regulatory elements responsible for early fruit development in cucurbit crops.

A number of cis-regulatory elements (CREs) conserved during evolution have been found to be responsible for phenotypic novelty and variation. Cucurbit crops such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), and squash (Cucurbita maxima) develop fruits from an inferior ovary and share some similar biological processes during fruit development. Whether conserved regulatory sequences play critical roles in fruit development of cucurbit crops remains to be explored. In six well-studied cucurbit species, we identified 392,438 conserved noncoding sequences (CNSs), including 82,756 that are specific to cucurbits, by comparative genomics. Genome-wide profiling of accessible chromatin regions (ACRs) and gene expression patterns mapped 20,865 to 43,204 ACRs and their potential target genes for two fruit tissues at two key developmental stages in six cucurbits. Integrated analysis of CNSs and ACRs revealed 4,431 syntenic orthologous CNSs, including 1,687 cucurbit-specific CNSs that overlap with ACRs that are present in all six cucurbit crops and that may regulate the expression of 757 adjacent orthologous genes. CRISPR mutations targeting two CNSs present in the 1,687 cucurbit-specific sequences resulted in substantially altered fruit shape and gene expression patterns of adjacent NAC1 (NAM, ATAF1/2, and CUC2) and EXT-like (EXTENSIN-like) genes, validating the regulatory roles of these CNSs in fruit development. These results not only provide a number of target CREs for cucurbit crop improvement, but also provide insight into the roles of CREs in plant biology and during evolution.

CmBr confers fruit bitterness under CPPU treatment in melon.

Many biotic or abiotic factors such as CPPU (N-(2-chloro-pyridin-4-yl)-N'-phenylurea), a growth regulator of numerous crops, can induce bitterness in cucurbits. In melon, cucurbitacin B is the major compound leading to bitterness. However, the molecular mechanism underlying CuB biosynthesis in response to different conditions remains unclear. Here, we identified a set of genes involved in CPPU-induced CuB biosynthesis in melon fruit and proposed CmBr gene as the major regulator. Using CRISPR/Cas9 gene editing, we confirmed CmBr's role in regulating CuB biosynthesis under CPPU treatment. We further discovered a CPPU-induced MYB-related transcription factor, CmRSM1, which specifically binds to the Myb motif within the CmBr promoter and activates its expression. Moreover, we developed an introgression line by introducing the mutated Cmbr gene into an elite variety and eliminated CPPU-induced bitterness, demonstrating its potential application in breeding. This study offers a valuable tool for breeding high-quality non-bitter melon varieties and provides new insights into the regulation of secondary metabolites under environmental stresses.

Novel Allelic Gene Variations in CmCLAVATA3 (CmCLV3) Were Identified in a Genetic Population of Melon (Cucumis melo L.).

Carpel number (CN) is an important trait affecting the fruit size and shape of melon, which plays a crucial role in determining the overall appearance and market value. A unique non-synonymous single nucleotide polymorphism (SNP) in CmCLAVATA3 (CmCLV3) is responsible for the variation of CN in C. melo ssp. agrestis (hereafter agrestis), but it has been unclear in C. melo ssp. melo (hereafter melo). In this study, one major locus controlling the polymorphism of 5-CN (multi-CN) and 3-CN (normal-CN) in melo was identified using bulked segregant analysis (BSA-seq). This locus was then fine-mapped to an interval of 1.8 Mb on chromosome 12 using a segregating population containing 1451 progeny. CmCLV3 is still present in the candidate region. A new allele of CmCLV3, which contains five other nucleotide polymorphisms, including a non-synonymous SNP in coding sequence (CDS), except the SNP reported in agrestis, was identified in melo. A cis-trans test confirmed that the candidate gene, CmCLV3, contributes to the variation of CNs in melo. The qRT-PCR results indicate that there is no significant difference in the expression level of CmCLV3 in the apical stem between the multi-CN plants and the normal-CN plants. Overall, this study provides a genetic resource for melon fruit development research and molecular breeding. Additionally, it suggests that melo has undergone similar genetic selection but evolved into an independent allele.

Arabidopsis Chloroplast protein for Growth and Fertility1 (CGF1) and CGF2 are essential for chloroplast development and female gametogenesis.

BACKGROUND: Chloroplasts are essential organelles of plant cells for not only being the energy factory but also making plant cells adaptable to different environmental stimuli. The nuclear genome encodes most of the chloroplast proteins, among which a large percentage of membrane proteins have yet to be functionally characterized. RESULTS: We report here functional characterization of two nuclear-encoded chloroplast proteins, Chloroplast protein for Growth and Fertility (CGF1) and CGF2. CGF1 and CGF2 are expressed in diverse tissues and developmental stages. Proteins they encode are associated with chloroplasts through a N-terminal chloroplast-targeting signal in green tissues but also located at plastids in roots and seeds. Mutants of CGF1 and CGF2 generated by CRISPR/Cas9 exhibited vegetative defects, including reduced leaf size, dwarfism, and abnormal cell death. CGF1 and CGF2 redundantly mediate female gametogenesis, likely by securing local energy supply. Indeed, mutations of both genes impaired chloroplast integrity whereas exogenous sucrose rescued the growth defects of the CGF double mutant. CONCLUSION: This study reports that two nuclear-encoded chloroplast proteins, Chloroplast protein for Growth and Fertility (CGF1) and CGF2, play important roles in vegetative growth, in female gametogenesis, and in embryogenesis likely by mediating chloroplast integrity and development.

Targeting and signaling of Rho of plants guanosine triphosphatases require synergistic interaction between guanine nucleotide inhibitor and vesicular trafficking.

Most eukaryotic cells are polarized. Common toolbox regulating cell polarization includes Rho guanosine triphosphatases (GTPases), in which spatiotemporal activation is regulated by a plethora of regulators. Rho of plants (ROPs) are the only Rho GTPases in plants. Although vesicular trafficking was hinted in the regulation of ROPs, it was unclear where vesicle-carried ROP starts, whether it is dynamically regulated, and which components participate in vesicle-mediated ROP targeting. In addition, although vesicle trafficking and guanine nucleotide inhibitor (GDI) pathways in Rho signaling have been extensively studied in yeast, it is unknown whether the two pathways interplay. Unclear are also cellular and developmental consequences of their interaction in multicellular organisms. Here, we show that the dynamic targeting of ROP through vesicles requires coat protein complex II and ADP-ribosylation factor 1-mediated post-Golgi trafficking. Trafficking of vesicle-carried ROPs between the plasma membrane and the trans-Golgi network is mediated through adaptor protein 1 and sterol-mediated endocytosis. Finally, we show that GDI and vesicle trafficking synergistically regulate cell polarization and ROP targeting, suggesting that the establishment and maintenance of cell polarity is regulated by an evolutionarily conserved mechanism.

S-acylation of CBL10/SCaBP8 by PAT10 is crucial for its tonoplast association and function in salt tolerance.

Crop yield is sensitive to salt stresses, for which Calcineurin B-like proteins (CBLs) are major response factors. This study shows that Arabidopsis CBL10, through protein S-acylation by protein S-acyl transferase10, targets to the vacuolar membrane to confer salt tolerance.

Arabidopsis PROTEIN S-ACYL TRANSFERASE4 mediates root hair growth.

Polar growth of root hairs is critical for plant survival and requires fine-tuned Rho of plants (ROP) signaling. Multiple ROP regulators participate in root hair growth. However, protein S-acyl transferases (PATs), mediating the S-acylation and membrane partitioning of ROPs, are yet to be found. Using a reverse genetic approach, combining fluorescence probes, pharmacological drugs, site-directed mutagenesis and genetic analysis with related root-hair mutants, we have identified and characterized an Arabidopsis PAT, which may be responsible for ROP2 S-acylation in root hairs. Specifically, functional loss of PAT4 resulted in reduced root hair elongation, which was rescued by a wild-type but not an enzyme-inactive PAT4. Membrane-associated ROP2 was significantly reduced in pat4, similar to S-acylation-deficient ROP2 in the wild type. We further showed that PAT4 and SCN1, a ROP regulator, additively mediate the stability and targeting of ROP2. The results presented here indicate that PAT4-mediated S-acylation mediates the membrane association of ROP2 at the root hair apex and provide novel insights into dynamic ROP signaling during plant tip growth.

PLURIPETALA mediates ROP2 localization and stability in parallel to SCN1 but synergistically with TIP1 in root hairs.

Prenylation, the post-translational attachment of prenyl groups to substrate proteins, can affect their distribution and interactomes. Arabidopsis PLURIPETALA (PLP) encodes the shared α subunit of two heterodimeric protein isoprenyltransferases, whose functional loss provides a unique opportunity to study developmental and cellular processes mediated by its prenylated substrates, such as ROP GTPases. As molecular switches, the distribution and activation of ROPs are mediated by various factors, including guanine nucleotide exchange factors, GTPase activating proteins, guanine nucleotide dissociation inhibitors (RhoGDIs), prenylation, and S-acylation. However, how these factors together ensure that dynamic ROP signalling is still obscure. We report here that a loss-of-function allele of PLP resulted in cytoplasmic accumulation of ROP2 in root hairs and reduced its stability. Consequently, two downstream events of ROP signalling, i.e. actin microfilament (MF) organization and the production of reactive oxygen species (ROS), were compromised. Genetic, cytological and biochemical evidence supports an additive interaction between prenylation and RhoGDI1/SCN1 in ROP2 distribution and stability whereas PLP acts synergistically with the protein S-acyl transferase TIP GROWTH DEFECTIVE1 during root hair growth. By using root hair growth as a model system, we uncovered complex interactions among prenylation, RhoGDIs, and S-acylation in dynamic ROP signalling.

Molecular cloning and characterization of gonadotropin subunits (GTHα, FSHß and LHß) and their regulation by hCG and GnRHa in Japanese sea bass (Lateolabrax japonicas) in vivo.

In this study, three cDNA sequences encoding common glycoprotein α subunit (GTHα), follicle-stimulating hormone ß subunit (FSHß) and luteinizing hormone ß subunit (LHß) were isolated from Japanese sea bass (Lateolabrax japonicas). Comparison of the deduced amino acid sequences with other gonadotropic hormones (GTHs) indicated that their cysteine residues and potential N-linked glycosylation sites were highly conserved, and high homology with those of other perciformes was showed in phylogenetic analysis. GTHs transcripts were present highly in the pituitary and brain and weakly in testis and other tissues. During testicular development, GTHs transcriptional levels in pituitary and brain (expect FSHß subunit in brain) were significantly increased at spermiation period, stage V. Subsequently, the effects of hCG and GnRHa on the mRNA levels of GTHs subunits were examined. In brain, both hormones were detected to improve the expression of GTHα subunit mRNA. In pituitary, three GTHs subunits increased parallelly and abruptly in two hormone treatment groups. In testis, hCG was suggested to improve three GTHs subunits expression in Japanese sea bass for the first time. These results suggest that both gonadotropins are probably involved in the control of Japanese sea bass spermatogenesis and provide a framework for better understanding of the mechanisms of hormone-mediated reproduction control in Japanese sea bass and other teleosts.

Life events and stress: do older men and women in Malaysia cope differently as consumers?

The study of major life events and their effects on well-being has considerable relevance for scientific disciplines and policy making in understanding the consumer behaviour of older people. There is evidence of differences in reactions to and coping with stress between males and females but relatively little knowledge about such gender differences amongst older people, especially in middle-income countries. This study of older Malaysians looked at both coping strategies and gender differences in reactions to stress when people are confronted with certain life events. Seventeen major life events were used in interviews with 645 respondents aged 50 years or older in five major urban areas in Peninsular Malaysia. The analysis showed older women tended to experience higher levels of chronic stress than older men. They also had more health problems, had lower levels of self-esteem and were less satisfied with life. Whilst the results showed little support for gender differences in coping behaviours, stress had a significant influence on the way older men and women change store preferences. A hypothesis that older women would use more emotion-focused coping strategies was not supported. Knowledge of how older Malaysians cope with life events and stress and especially in this instance with regard to consumption behaviour, is likely to be of considerable academic and policy related interest.

Gibberellin biosynthesis is required for CPPU-induced parthenocarpy in melon.

Spraying N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU), an exogenous cytokinin (CK) growth regulator, is the conventional method for inducing fruit set during melon (Cucumis melo L.) production; however, the mechanism by which CPPU induces fruit set is unclear. Through histological and morphological observations, fruit size was comparable between CPPU-induced fruits and normal pollinated fruits because CPPU-induced fruits had higher cell density but smaller cell size compared with normal pollinated fruits. CPPU promotes the accumulation of gibberellin (GA) and auxin and decreases the level of abscisic acid (ABA) during fruit set. Moreover, application of the GA inhibitor paclobutrazol (PAC) partially inhibits CPPU-induced fruit set. Transcriptome analysis revealed that CPPU-induced fruit set specifically induced the GA-related pathway, in which the key synthase encoding gibberellin 20-oxidase 1 (CmGA20ox1) was specifically upregulated. Further study indicated that the two-component response regulator 2 (CmRR2) of the cytokinin signaling pathway, which is highly expressed at fruit setting, positively regulates the expression of CmGA20ox1. Collectively, our study determined that CPPU-induced melon fruit set is dependent on GA biosynthesis, providing a theoretical basis for the creation of parthenocarpic melon germplasm.

Distributed Model-Free Sliding-Mode Predictive Control of Discrete-Time Second-Order Nonlinear Multiagent Systems With Delays.

In this article, the tracking problem of networked discrete-time second-order nonlinear multiagent systems (MASs) is studied. First, for the MASs without communication delay, a novel method, called distributed model-free sliding-mode control algorithm is proposed, which can make the system converge quickly without the accurate model. Furthermore, for the MASs with delay, in order to eliminate the influence of time delay on the system, a distributed model-free sliding-mode predictive control strategy based on time-delay compensation technology is proposed, which can actively compensate for time delay while ensuring system stability and consensus tracking performance requirements. Both the simulation and experiment results reveal the superiority of the proposed methods.

Comparative genomics analysis of bHLH genes in cucurbits identifies a novel gene regulating cucurbitacin biosynthesis.

The basic helix-loop-helix (bHLH) family of transcription factors (TFs) participate in a variety of biological regulatory processes in plants, and have undergone significant expansion during land plant evolution by gene duplications. In cucurbit crops, several bHLH genes have been found to be responsible for the agronomic traits such as bitterness. However, the characterization of bHLH genes across the genomes of cucurbit species has not been reported, and how they have evolved and diverged remains largely unanswered. Here we identified 1160 bHLH genes in seven cucurbit crops and performed a comprehensive comparative genomics analysis. We determined orthologous and paralogous bHLH genes across cucurbit crops by syntenic analysis between or within species. Orthology and phylogenetic analysis of the tandem-duplicated bHLH genes in the Bt cluster which regulate the biosynthesis of cucurbitacins suggest that this cluster is derived from three ancestral genes after the cucurbit-common tetraploidization event. Interestingly, we identified a new conserved cluster paralogous to the Bt cluster that includes two tandem bHLH genes, and the evolutionary history and expression profiles of these two genes in the new cluster suggest the involvement of one gene (Brp) in the regulation of cucurbitacin biosynthesis in roots. Further biochemical and transgenic assays in melon hairy roots support the function of Brp. This study provides useful information for further investigating the functions of bHLH TFs and novel insights into the regulation of cucurbitacin biosynthesis in cucurbit crops and other plants.

Graph-based pan-genome reveals structural and sequence variations related to agronomic traits and domestication in cucumber.

Structural variants (SVs) represent a major source of genetic diversity and are related to numerous agronomic traits and evolutionary events; however, their comprehensive identification and characterization in cucumber (Cucumis sativus L.) have been hindered by the lack of a high-quality pan-genome. Here, we report a graph-based cucumber pan-genome by analyzing twelve chromosome-scale genome assemblies. Genotyping of seven large chromosomal rearrangements based on the pan-genome provides useful information for use of wild accessions in breeding and genetic studies. A total of ~4.3 million genetic variants including 56,214 SVs are identified leveraging the chromosome-level assemblies. The pan-genome graph integrating both variant information and reference genome sequences aids the identification of SVs associated with agronomic traits, including warty fruits, flowering times and root growth, and enhances the understanding of cucumber trait evolution. The graph-based cucumber pan-genome and the identified genetic variants provide rich resources for future biological research and genomics-assisted breeding.

Sugars promote graft union development in the heterograft of cucumber onto pumpkin.

The use of heterografts is widely applied for the production of several important commercial crops, but the molecular mechanism of graft union formation remains poorly understood. Here, cucumber grafted onto pumpkin was used to study graft union development, and genome-wide tempo-spatial gene expression at the graft interface was comprehensively investigated. Histological analysis suggested that resumption of the rootstock growth occurred after both phloem and xylem reconnection, and the scion showed evident callus production compared with the rootstock 3 days after grafting. Consistently, transcriptome data revealed specific responses between the scion and rootstock in the expression of genes related to cambium development, the cell cycle, and sugar metabolism during both vascular reconnection and healing, indicating distinct mechanisms. Additionally, lower levels of sugars and significantly changed sugar enzyme activities at the graft junction were observed during vascular reconnection. Next, we found that the healing process of grafted etiolated seedlings was significantly delayed, and graft success, xylem reconnection, and the growth of grafted plants were enhanced by exogenous glucose. This demonstrates that graft union formation requires the correct sugar content. Furthermore, we also found that graft union formation was delayed with a lower energy charge by the target of rapamycin (TOR) inhibitor AZD-8055, and xylem reconnection and the growth of grafted plants were enhanced under AZD-8055 with exogenous glucose treatment. Taken together, our results reveal that sugars play a positive role in graft union formation by promoting the growth of cucumber/pumpkin and provide useful information for understanding graft union healing and the application of heterografting in the future.

Nitrate deficiency induces differential endocytosis in roots through NRT1.1.

Roots grow asymmetrically, sometimes helically, around their growth direction likely to facilitate environmental sensing. We recently demonstrated that nitrate deficiency induces root coiling on horizontal surface through nitrate transporter/sensor NRT1.1 and PIN2- and AUX-mediated polar auxin transport. Here, we show that nitrate deficiency or NRT1.1 loss-of-function induces differential distribution of PIN2 between the future concave and concave sides in root epidermal cells. Treatment with pharmacological drugs suggests that enhanced endocytosis at the future convex side leads to reduced plasma membrane (PM) association of PIN2. A reduction of PIN2 at the PM would maintain a low auxin response to further enhance endocytosis at the convex side, leading to root coiling.

NRT1.1-Mediated Nitrate Suppression of Root Coiling Relies on PIN2- and AUX1-Mediated Auxin Transport.

Asymmetric root growth (ARG) on tilted plates, or root coiling on horizontally placed plates, is proposed to be a combination of gravitropism, mechanical sensing, and "circumnutation," a word designated by Charles Darwin to describe the helical movement that all plant organs make around the growth direction. ARG is developmentally controlled in which microtubule-regulating proteins and the phytohormone auxin participates. Nutrient deficiency influences ARG. However, it is unclear which nutrients play key roles in regulating ARG, what endogenous components are involved in responding to nutrient deficiency for ARG, and how nutrient deficiency is translated into endogenous responses. We report here that nitrate deficiency resulted in a strong ARG in Arabidopsis. Nitrate deficiency caused root coiling on horizontal plates, which is inhibited by an auxin transport inhibitor, and by mutations in PIN-FORMED2 (PIN2) and AUXIN RESISTANT 1 (AUX1). We further show that suppression of ARG by nitrate is mediated by the nitrate transporter/sensor NRT1.1. In addition, PIN2- and AUX1-mediated auxin transports are epistatic to NRT1.1 in nitrate deficiency-induced ARG. This study reveals a signaling pathway in root growth by responding to exogenous nitrate and relaying it into altered auxin transport.

HUA ENHANCER1 Mediates Ovule Development.

Ovules are female reproductive organs of angiosperms, containing sporophytic integuments and gametophytic embryo sacs. After fertilization, embryo sacs develop into embryos and endosperm whereas integuments into seed coat. Ovule development is regulated by transcription factors (TF) whose expression is often controlled by microRNAs. Mutations of Arabidopsis DICER-LIKE 1 (DCL1), a microRNA processing protein, caused defective ovule development and reduced female fertility. However, it was not clear whether other microRNA processing proteins participate in this process and how defective ovule development influenced female fertility. We report that mutations of HUA ENHANCER1 (HEN1) and HYPONASTIC LEAVES 1 (HYL1) interfered with integument growth. The sporophytic defect caused abnormal embryo sac development and inability of mutant ovules to attract pollen tubes, leading to reduced female fertility. We show that the role of HEN1 in integument growth is cell-autonomous. Although AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 were ectopically expressed in mutant ovules, consistent with the reduction of microRNA167 in hen1, introducing arf6;arf8 did not suppress ovule defects of hen1, suggesting the involvement of more microRNAs in this process. Results presented indicate that the microRNA processing machinery is critical for ovule development and seed production through multiple microRNAs and their targets.

Effect of dry heating treatment on multi-levels of structure and physicochemical properties of maize starch: A thermodynamic study.

Dry heating treatment (DHT) is a common process widely used in food industry. In this study, the thermodynamic effects of DHT on starch structure and physicochemical properties are investigated. The results show that, with heating temperature increasing during DHT, the molecular size, long-amylose chains with degree of polymerization (DP) ~5000-20,000, and the crystallinity of maize starch are significantly reduced while the granular structure is retained with slightly aggregation between starch particles. The solubility of DHT starch increases from 0.5% to 2%, indicating the majority of DHT starch is still insoluble. DHT affects starch thermal property greatly that, it decreases the gelatinization enthalpy while increases the heterogeneity of starch crystallites. With heating temperature increasing, DHT reduces the overall viscosity of starch paste. The rheological property of DHT starch is frequency-dependent, showing the typical shear thinning behavior and "solid-like" gel property. Especially, as heating temperature reaches 190 °C, the shear resistance becomes stronger, and it is closer to Newtonian fluid. The results prove the thermodynamic effects of DHT on multi-levels of starch structure and physicochemical properties, also indicating the great potential to utilize DHT in modifying starch properties and amplifying its applications.

AGC1.5 Kinase Phosphorylates RopGEFs to Control Pollen Tube Growth.

Double fertilization in angiosperms requires the targeted delivery of immotile sperm to the eggs through pollen tubes. The polarity of tip-growing pollen tubes is maintained through dynamic association of active Rho GTPases of plants (ROP-GTP) with the apical plasma membrane. Guanine nucleotide exchange factors for ROPs (RopGEFs) catalyze the activation of ROPs and thereby affect spatiotemporal ROP signaling. Whereas RopGEFs have been found to be phosphorylated proteins, the kinases responsible for their phosphorylation in vivo and biological consequences of RopGEF phosphorylation in pollen tube growth remain unclear. We report here that the Arabidopsis AGC1.5 subfamily of cytoplasmic kinases is critical for the restricted localization of ROP-GTP during pollen tube growth. Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of active ROPs and defective events downstream of ROP signaling in pollen tubes. AGC1.5 interacts with RopGEFs via their catalytic PRONE domain and phosphorylates RopGEFs at a conserved Ser residue of PRONE domain. Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of RopGEFs in pollen tubes, similar to the phenotype caused by the mutation that renders RopGEFs non-phosphorylatable by AGC1.5. Collectively, our results provide mechanistic insights into the spatiotemporal activation of ROPs during the polar growth of pollen tubes.