Integrated transcriptomics and metabolomics provides insights into the Nicotiana tabacum response to heat stress.

Heat stress is a prevalent factor that significantly damages crops, especially with the ongoing global warming and increasing frequency of extreme weather events. Tobacco is particularly sensitive to temperature fluctuations, experiencing reduced yield and quality under high temperatures. However, the underlying molecular mechanisms of heat resistance in tobacco remain poorly understood. This study comprehensively analyzed biochemical, transcriptomic, and metabolomic responses to heat stress on the root and shoot of the tobacco cultivar K326 compared to control conditions. Heat stress significantly increased the activities of antioxidant enzymes (CAT, POD, and SOD) and levels of osmotic mediators (soluble sugars, sucrose, and proline) in the shoot. Furthermore, transcriptome analysis identified 13,176 differentially expressed genes (DEGs) in the root (6,129 up-regulated and 7,047 down-regulated) and 12,283 DEGs (6,621 up-regulated and 5,662 down-regulated) in the shoot. The root had 24 enriched KEGG pathways, including phenylpropanoid metabolism, while the shoot had 32 significant pathways, such as galactose metabolism and MAPK signaling. The metabolomic data identified 647 metabolites in the root and 932 in the shoot, with carbohydrates and amino acids being the main categories. The root had 116 differentially abundant metabolites (DAMs) (107 up-regulated and 9 down-regulated), and the shoot contained 256 DAMs (251 up-regulated and 5 down-regulated). Joint transcriptome and metabolome analysis showed that galactose metabolism and starch and sucrose metabolism were co-enriched in both tissues. In contrast, amino sugar and nucleotide sugar metabolism was enriched in the root, and purine metabolism in the shoot. The purine metabolic pathway in the shoot can modulate the expression of MYB transcription factors by influencing ABA synthesis and signaling, thereby controlling the accumulation of HSPs, raffinose, sucrose, and trehalose to enhance heat tolerance. Furthermore, NtMYB78, an MYB transcription factor, enhances tolerance for heat stress in tobacco. This research offers a foundational framework for investigating and implementing heat-resistant genes and metabolic pathways in the root and shoot of tobacco seedlings.

Integrative proteome and metabolome unveil the central role of IAA alteration in axillary bud development following topping in tobacco.

Axillary bud is an important aspect of plant morphology, contributing to the final tobacco yield. However, the mechanisms of axillary bud development in tobacco remain largely unknown. To investigate this aspect of tobacco biology, the metabolome and proteome of the axillary buds before and after topping were compared. A total of 569 metabolites were differentially abundant before and 1, 3, and 5 days after topping. KEGG analyses further revealed that the axillary bud was characterized by a striking enrichment of metabolites involved in flavonoid metabolism, suggesting a strong flavonoid biosynthesis activity in the tobacco axillary bud after topping. Additionally, 9035 differentially expressed proteins (DEPs) were identified before and 1, 3, and 5 days after topping. Subsequent GO and KEGG analyses revealed that the DEPs in the axillary bud were enriched in oxidative stress, hormone signal transduction, MAPK signaling pathway, and starch and sucrose metabolism. The integrated proteome and metabolome analysis revealed that the indole-3-acetic acid (IAA) alteration in buds control dormancy release and sustained growth of axillary bud by regulating proteins involved in carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Notably, the proteins related to reactive oxygen species (ROS) scavenging and flavonoid biosynthesis were strongly negatively correlated with IAA content. These findings shed light on a critical role of IAA alteration in regulating axillary bud outgrowth, and implied a potential crosstalk among IAA alteration, ROS homeostasis, and flavonoid biosynthesis in tobacco axillary bud under topping stress, which could improve our understanding of the IAA alteration in axillary bud as an important regulator of axillary bud development.

Chilling stress response in tobacco seedlings: insights from transcriptome, proteome, and phosphoproteome analyses.

Tobacco (Nicotiana tabacum L.) is an important industrial crop, which is sensitive to chilling stress. Tobacco seedlings that have been subjected to chilling stress readily flower early, which seriously affects the yield and quality of their leaves. Currently, there has been progress in elucidating the molecular mechanisms by which tobacco responds to chilling stress. However, little is known about the phosphorylation that is mediated by chilling. In this study, the transcriptome, proteome and phosphoproteome were analyzed to elucidate the mechanisms of the responses of tobacco shoot and root to chilling stress (4 °C for 24 h). A total of 6,113 differentially expressed genes (DEGs), 153 differentially expressed proteins (DEPs) and 345 differential phosphopeptides were identified in the shoot, and the corresponding numbers in the root were 6,394, 212 and 404, respectively. This study showed that the tobacco seedlings to 24 h of chilling stress primarily responded to this phenomenon by altering their levels of phosphopeptide abundance. Kyoto Encyclopedia of Genes and Genomes analyses revealed that starch and sucrose metabolism and endocytosis were the common pathways in the shoot and root at these levels. In addition, the differential phosphopeptide corresponding proteins were also significantly enriched in the pathways of photosynthesis-antenna proteins and carbon fixation in photosynthetic organisms in the shoot and arginine and proline metabolism, peroxisome and RNA transport in the root. These results suggest that phosphoproteins in these pathways play important roles in the response to chilling stress. Moreover, kinases and transcription factors (TFs) that respond to chilling at the levels of phosphorylation are also crucial for resistance to chilling in tobacco seedlings. The phosphorylation or dephosphorylation of kinases, such as CDPKs and RLKs; and TFs, including VIP1-like, ABI5-like protein 2, TCP7-like, WRKY 6-like, MYC2-like and CAMTA7 among others, may play essential roles in the transduction of tobacco chilling signal and the transcriptional regulation of the genes that respond to chilling stress. Taken together, these findings provide new insights into the molecular mechanisms and regulatory networks of the responses of tobacco to chilling stress.

Farnesoid X receptor represses human sulfotransferase 1A3 expression through direct binding to the promoter.

Human sulfotransferases 1A3 (SULT1A3) has received particular interest, due to their functions of catalyzing the sulfonation of numerous phenolic substrates, including bioactive endogenous molecules and therapeutic agents. However, the regulation of SULT1A3 expression and the underlying mechanism remain unclear. Here, we aimed to investigate the regulation effects of bile acid-activated farnesoid X receptor (FXR) on SULT1A3 expression, and to shed light on the mechanism thereof. Our results demonstrated that FXR agonists (CDCA and GW4064) significantly inhibit the expression of SULT1A3 at mRNA and protein levels. In addition, overexpression of FXR led to decrease in SULT1A3 expression and knockdown of FXR significantly induced the expression of SULT1A3 in protein and mRNA levels, confirming that FXR expression manifestly showed negative regulatory effect on basal SULT1A3 expression. Furthermore, a combination of luciferase reporter gene and CHIP assays showed that FXR repressed SULT1A3 transcription through direct binding to the region at base pair positions -664 to -654. In conclusion, this study for the first time confirmed FXR was a negative transcriptional regulator of human SULT1A3 enzyme.

Multi-Objective Optimization of Sugarcane Milling System Operations Based on a Deep Data-Driven Model.

The extraction of sugarcane juice is the first step of sugar production. The optimal values of process indicators and the set values of operating parameters in this process are still determined by workers' experience, preventing adaptive adjustment of the production process. To address this issue, a multi-objective optimization framework based on a deep data-driven model is proposed to optimize the operation of sugarcane milling systems. First, the sugarcane milling process is abstracted as the interaction of material flow, energy flow, and information flow (MF-EF-IF) by introducing synergetic theory, and each flow's order parameters and state parameters are obtained. Subsequently, the state parameters of the subsystems are taken as inputs, and the order parameters-including the grinding capacity, electric consumption per ton of sugarcane, and sucrose extraction-are produced as outputs. A collaborative optimization model of the MF-EF-IF of the milling system is established by using a deep kernel extreme learning machine (DK-ELM). The established milling system model is applied for an improved multi-objective chicken swarm optimization (IMOCSO) algorithm to obtain the optimal values of the order parameters. Finally, the milling process is described as a Markov decision process (MDP) with the optimal values of the order parameters as the control objectives, and an improved deep deterministic policy gradient (DDPG) algorithm is employed to achieve the adaptive optimization of the operating parameters under different working conditions of the milling system. Computational experiments indicate that enhanced performance is achieved, with an increase of 3.2 t per hour in grinding capacity, a reduction of 660 W per ton in sugarcane electric consumption, and an increase of 0.03% in the sucrose extraction.

Maize VKS1 Regulates Mitosis and Cytokinesis During Early Endosperm Development.

Cell number is a critical factor that determines kernel size in maize (Zea mays). Rapid mitotic divisions in early endosperm development produce most of the cells that make up the starchy endosperm; however, the mechanisms underlying early endosperm development remain largely unknown. We isolated a maize mutant that shows a varied-kernel-size phenotype (vks1). Vks1 encodes ZmKIN11, which belongs to the kinesin-14 subfamily and is predominantly expressed in early endosperm development. VKS1 dynamically localizes to the nucleus and microtubules and plays key roles in the migration of free nuclei in the coenocyte as well as in mitosis and cytokinesis in early mitotic divisions. Absence of VKS1 has relatively minor effects on plants but causes deformities in spindle assembly, sister chromatid separation, and phragmoplast formation in early endosperm development, thereby resulting in reduced cell proliferation. Severities of aberrant mitosis and cytokinesis within individual vks1 endosperms differ, thereby resulting in varied kernel sizes. Our discovery highlights VKS1 as a central regulator of mitosis in early maize endosperm development and provides a potential approach for future yield improvement.

Phase-Field Modeling of Freeze Concentration of Protein Solutions.

Bulk solutions of therapeutic proteins are often frozen for long-term storage. During the freezing process, proteins in liquid solution redistribute and segregate in the interstitial space between ice crystals. This is due to solute exclusion from ice crystals, higher viscosity of the concentrated solution, and space confinement between crystals. Such segregation may have a negative impact on the native conformation of protein molecules. To better understand the mechanisms, we developed a phase-field model to describe the growth of ice crystals and the dynamics of freeze concentration at the mesoscale based on mean field approximation of solute concentration and the underlying heat, mass and momentum transport phenomena. The model focuses on evolution of the interfaces between liquid solution and ice crystals, and the degree of solute concentration due to partition, diffusive, and convective effects. The growth of crystals is driven by cooling of the bulk solution, but suppressed by a higher solute concentration due to increase of solution viscosity, decrease of freezing point, and the release of latent heat. The results demonstrate the interplay of solute exclusion, space confinement, heat transfer, coalescence of crystals, and the dynamic formation of narrow gaps between crystals and Plateau border areas along with correlations of thermophysical properties in the supercooled regime.

Integrated Experimental and Modeling Study of Enzymatic Degradation Using Novel Autofluorescent BSA Microspheres.

Autofluorescent bovine serum albumin (BSA) hydrogel microspheres were prepared through the spray-drying of glutaraldehyde cross-linked BSA nanoparticles and then used for a proteinase K based degradation study in an aqueous solution. Experimental results and empirical models are presented to characterize the kinetics of BSA hydrogel microsphere degradation, as well as the accompanying release of synthesized fluorophore. The BSA gel degradation dynamics is primarily controlled by the concentration of proteinase K within the Tris buffer. The coupling of swelling dynamics and the transient distributions of fluorophore are traced by confocal microscopy. Models are developed based on the linear theory of elastic deformation coupled to enzyme and fluorophore transport. This study represents a fundamental investigation of the degradation and release kinetics of protein-based materials, which can potentially be applied for the dynamic and photostable tracking of relevant in vivo systems.

Divergent regeneration-competent cells adopt a common mechanism for callus initiation in angiosperms.

In tissue culture, the formation of callus from detached explants is a key step in plant regeneration; however, the regenerative abilities in different species are variable. While nearly all parts of organs of the dicot Arabidopsis thaliana are ready for callus formation, mature regions of organs in monocot rice (Oryza sativa) and other cereals are extremely unresponsive to tissue culture. Whether there is a common molecular mechanism beyond these different regenerative phenomena is unclear. Here we show that the Arabidopsis and rice use different regeneration-competent cells to initiate callus, whereas the cells all adopt WUSCHEL-RELATED HOMEOBOX 11 (WOX11) and WOX5 during cell fate transition. Different from Arabidopsis which maintains regeneration-competent cells in mature organs, rice exhausts those cells during organ maturation, resulting in regenerative inability in mature organs. Our study not only explains this old perplexity in agricultural biotechnology, but also provides common molecular markers for tissue culture of different angiosperm species.

WOX11 and 12 are involved in the first-step cell fate transition during de novo root organogenesis in Arabidopsis.

De novo organogenesis is a process through which wounded or detached plant tissues or organs regenerate adventitious roots and shoots. Plant hormones play key roles in de novo organogenesis, whereas the mechanism by which hormonal actions result in the first-step cell fate transition in the whole process is unknown. Using leaf explants of Arabidopsis thaliana, we show that the homeobox genes WUSCHEL RELATED HOMEOBOX11 (WOX11) and WOX12 are involved in de novo root organogenesis. WOX11 directly responds to a wounding-induced auxin maximum in and surrounding the procambium and acts redundantly with its homolog WOX12 to upregulate LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) and LBD29, resulting in the first-step cell fate transition from a leaf procambium or its nearby parenchyma cell to a root founder cell. In addition, our results suggest that de novo root organogenesis and callus formation share a similar mechanism at initiation.

Quantitative control of ASYMMETRIC LEAVES2 expression is critical for leaf axial patterning in Arabidopsis.

ASYMMETRIC LEAVES2 (AS2) is one of the key genes required for specifying leaf adaxial identity during leaf adaxial-abaxial polarity establishment. Previous data have shown that, in leaf development, AS2 is directly repressed by an abaxially located transcription factor KANADI1 (KAN1), so that the AS2 transcripts are restricted only in the adaxial leaf domain. It is shown here that, different from the spatial repression by KAN1, the quantitative repression of AS2 in the adaxial domain is also critical for ensuring normal leaf pattern formation. By analysing two gain-of-function as2 mutants, as2-5D and isoginchaku-2D (iso-2D), it is shown that the similar AS2-over-expressed phenotypes of these mutants reflect two different kinds of AS2 misexpression patterns. While as2-5D causes disruption of a KAN1-binding site at the AS2 promoter leading to derepression of AS2 in the abaxial side but without changing its expression level of a leaf, iso-2D results in over-expression of AS2 but without altering its adaxial expression pattern. In addition, it was found that, in iso-2D, levels of histone H3 lysine 27 trimethylation (H3K27me3) and H3K4me3 at the AS2 locus are significantly reduced and increased, respectively, compared with those in the wild type and as2-5D. These results suggest that during leaf patterning, quantitative control of the AS2 expression level might involve epigenetic regulations.

Molecular characterization of TGF-ß type I receptor gene (Tgfbr1) in Chlamys farreri, and the association of allelic variants with growth traits.

BACKGROUND: Scallops are an economically important aquaculture species in Asian countries, and growth-rate improvement is one of the main focuses of scallop breeding. Investigating the genetic regulation of scallop growth could benefit scallop breeding, as such research is currently limited. The transforming growth factor beta (TGF-ß) signaling through type I and type II receptors, plays critical roles in regulating cell proliferation and growth, and is thus a plausible candidate growth regulator in scallops. RESULTS: We cloned and characterized the TGF-ß type I receptor (Tgfbr1) gene from Zhikong scallops (Chlamys farreri). The deduced amino acid sequence contains characteristic residues and exhibits the conserved structure of Tgfbr1 proteins. A high expression level of scallop Tgfbr1 was detected during early embryonic stages, whereas Tgfbr1 expression was enriched in the gonad and striated muscle in adults. A single nucleotide polymorphism (SNP, c. 1815C>T) in the 3' UTR was identified. Scallops with genotype TT had higher growth traits values than those with genotype CC or CT in a full-sib family, and significant differences were found between genotypes CC and TT for shell length, shell height, and striated muscle weight. An expression analysis detected significantly more Tgfbr1 transcripts in the striated muscle of scallops with genotype CC compared to those with genotype TT or CT. Further evaluation in a population also revealed higher striated muscle weight in scallops with genotype TT than those with the other two genotypes. The inverse correlation between striated muscle mass and Tgfbr1 expression is consistent with TGF-ß signaling having a negative effect on cell growth. CONCLUSION: The scallop Tgfbr1 gene was cloned and characterized, and an SNP potentially associated with both scallop growth and Tgfbr1 expression was identified. Our results suggest the negative regulation of Tgfbr1 in scallop growth and provide a candidate marker for Zhikong scallop breeding.

Imitation Switch chromatin remodeling factors and their interacting RINGLET proteins act together in controlling the plant vegetative phase in Arabidopsis.

During their life cycle, flowering plants must experience a transition from vegetative to reproductive growth. Here, we report that double mutations in the Arabidopsis thaliana IMITATION SWITCH (AtISWI) genes, CHROMATIN REMODELING11 (CHR11) and CHR17, and the plant-specific DDT-domain containing genes, RINGLET1 (RLT1) and RLT2, resulted in plants with similar developmental defects, including the dramatically accelerated vegetative-to-reproductive transition. We demonstrated that AtISWI physically interacts with RLTs in preventing plants from activating the vegetative-to-reproductive transition early by regulating several key genes that contribute to flower timing. In particular, AtISWI and RLTs repress FT, SEP1, SEP3, FUL, and SOC1, but promote FLC in the leaf. Furthermore, AtISWI and RLTs may directly repress FT and SEP3 through associating with the FT and SEP3 loci. Our study reveals that AtISWI and RLTs represent a previously unrecognized genetic pathway that is required for the maintenance of the plant vegetative phase.

YUCCA genes are expressed in response to leaf adaxial-abaxial juxtaposition and are required for leaf margin development.

During leaf development, the formation of leaf adaxial-abaxial polarity at the primordium stage is crucial for subsequent leaf expansion. However, little is known about the genetic control from polarity establishment to blade outgrowth. The leaf margin, comprising elongated margin cells and hydathodes, is thought to affect leaf expansion. Here, we show that mutants with defective leaf polarity or with loss of function in the multiple auxin-biosynthetic YUCCA (YUC) genes exhibited a similar abnormal leaf margin and less-expanded leaves. Leaf margins of these mutants contained fewer hydathodes and an increased number of cell patches in which the patterns of epidermal cells resembled those of hydathodes. The previously characterized leaf-abaxialized asymmetric leaves2 (as2) revoluta (rev) and leaf-adaxialized kanadi1 (kan1) kan2 double mutants both produce finger-shaped, hydathode-like protrusions on adaxial and abaxial leaf surfaces, respectively. YUCs are required for formation of the protrusions, as those produced by as2 rev and kan1 kan2 were absent in the yuc1 yuc2 yuc4 triple mutant background. Expressions of YUC1, YUC2, and YUC4 were spatially regulated in the leaf, being associated with hydathodes in wild-type leaves and protrusions on as2 rev and kan1 kan2 leaves. In addition, inhibition of auxin transport by treatment of seedlings with N-(1-naphtyl) phtalamic acid or disruption of the auxin gradient by transforming plants with the 35S:YUC1 construct also blocked leaf margin development. Collectively, our data show that expressions of YUCs in the leaf respond to the adaxial-abaxial juxtaposition, and that the activities of auxin mediate leaf margin development, which subsequently promotes blade outgrowth.