Root restriction is an agronomic technique that influences plant morphology, physiology, and productivity. This study investigates the effects of root restriction on tomato growth, fruit quality, yield, and rhizosphere microbial communities using three distinct substrates: sand, soil, and peanut shell substrate (PSS), within a Simplified Automatic Soilless Culture System (SAS). Results demonstrated that root restriction at 8 cm height significantly enhanced fruit quality indicators: soluble sugar content increased by 69.01% (sand), 53.84% (soil), and 37.67% (PSS); soluble protein increased by 77.23%, 48.14%, and 66.51%; and lycopene increased by 100.03%, 62.33%, and 74.59%, respectively, compared to the 24 cm baseline. However, single-plant yield declined by 28.30% (sand), 64.28% (soil), and 22.06% (PSS). TOPSIS analysis (Technique for Order Preference by Similarity to Ideal Solution) identified PSS at 8 cm as the optimal combination for balancing quality and yield (Cj = 0.631). Microbial amplicon sequencing revealed higher rhizosphere microbial diversity in tomatoes grown in soil and peanut shell substrate compared to sand. These three types of growing media (soil, sand, and peanut shell substrate) establish the rhizosphere of bacterial and fungal communities by selecting specific microbial taxa. Changes in container height drive the reduction-oxidation functional divergence of bacterial communities, affecting the connectivity and complexity of microbial networks.

Arbuscular mycorrhizal symbiosis (AMS) is a ubiquitous mutualistic interaction between many terrestrial plants and fungi, with lipids playing a pivotal role in nutrient exchange. However, few genetic regulators of AMS have been functionally validated in tomato. To investigate candidate genes, we employed CRISPR‐Cas9 and VIGS to generate knockout and knockdown lines. A comprehensive suite of molecular biology techniques, including yeast‐1/2‐hybridization, BiFC, ChIP‐qPCR, and RNA‐sequencing, was used to elucidate the regulatory roles of SlWRI5a, SlHY5, and SlFatM in fatty acid (FA) biosynthesis and AMS in tomato. FA composition was analyzed using gas chromatography. In this study, we validated SlWRI5a and SlFatM as key regulators of 16‐carbon FA biosynthesis during AMS in tomato and demonstrated physical interactions between SlWRI5a and SlHY5. SlHY5 expression was induced by AMS and promoted root FA biosynthesis. Finally, we demonstrated that SlWRI5a and SlHY5 can co‐regulate SlFatM‐mediated FA accumulation, thereby influencing AMF colonization efficiency in tomato. Our findings reveal the SlWRI5a/SlHY5–SlFatM regulatory module, offering new insights into lipid‐mediated AMS in tomato. This work also highlights a novel role for HY5 during fungal symbiosis, underscoring its broader significance in plant–microbe interactions.

Cold stress is a major environmental challenge affecting the production of crops. Calcium‐dependent protein kinases (CDPKs/CPKs) are crucial regulators relaying calcium (Ca 2+ ) signals into cellular stress responses. However, the specific mechanisms of CPKs in regulating cold stress signaling are not well understood. In this study, through genetic, physiological and molecular biology assays, we characterized the function of CPK27 in enhancing tomato cold tolerance. We found that CPK27 stimulates flavonoid biosynthesis in a Ca 2+ ‐dependent manner, which in turn boosts the plant's tolerance. Tomato plants lacking CPK27 ( cpk27 ) showed decreased flavonoid levels under cold stress, accompanied by the increased sensitivity to cold. Activated by cold stress, CPK27 accumulates within the nucleus, where it physically interacts and phosphorylates ELONGATED HYPOCOTYL 5 (HY5) protein at serine23 (S23) and S57 residues, contributing to the cold‐induced accumulation of HY5 protein. HY5 directly binds to the promoter regions and stimulates the transcription of flavonoid biosynthesis genes. Further genetic analysis showed that CPK27 acts upstream of HY5, and the flavonoid biosynthesis pathway activated by CPK27 is HY5‐dependent. Our study elucidates the regulatory mechanism whereby the CPK27‐HY5 molecule integrates cold‐triggered Ca 2+ signals with flavonoid biosynthesis pathways to confer cold stress tolerance, thereby uncovering the key strategy for cold signal transduction.

Arbuscular mycorrhizal symbiosis (AMS) is a ubiquitous mutualistic interaction between many terrestrial plants and fungi, with lipids playing a pivotal role in nutrient exchange. However, few genetic regulators of AMS have been functionally validated in tomato. To investigate candidate genes, we employed CRISPR-Cas9 and VIGS to generate knockout and knockdown lines. A comprehensive suite of molecular biology techniques, including yeast-1/2-hybridization, BiFC, ChIP-qPCR, and RNA-sequencing, was used to elucidate the regulatory roles of SlWRI5a, SlHY5, and SlFatM in fatty acid (FA) biosynthesis and AMS in tomato. FA composition was analyzed using gas chromatography. In this study, we validated SlWRI5a and SlFatM as key regulators of 16-carbon FA biosynthesis during AMS in tomato and demonstrated physical interactions between SlWRI5a and SlHY5. SlHY5 expression was induced by AMS and promoted root FA biosynthesis. Finally, we demonstrated that SlWRI5a and SlHY5 can co-regulate SlFatM-mediated FA accumulation, thereby influencing AMF colonization efficiency in tomato. Our findings reveal the SlWRI5a/SlHY5-SlFatM regulatory module, offering new insights into lipid-mediated AMS in tomato. This work also highlights a novel role for HY5 during fungal symbiosis, underscoring its broader significance in plant-microbe interactions.

Cold stress is a major environmental challenge affecting the production of crops. Calcium-dependent protein kinases (CDPKs/CPKs) are crucial regulators relaying calcium (Ca2+) signals into cellular stress responses. However, the specific mechanisms of CPKs in regulating cold stress signaling are not well understood. In this study, through genetic, physiological and molecular biology assays, we characterized the function of CPK27 in enhancing tomato cold tolerance. We found that CPK27 stimulates flavonoid biosynthesis in a Ca2+-dependent manner, which in turn boosts the plant's tolerance. Tomato plants lacking CPK27 (cpk27) showed decreased flavonoid levels under cold stress, accompanied by the increased sensitivity to cold. Activated by cold stress, CPK27 accumulates within the nucleus, where it physically interacts and phosphorylates ELONGATED HYPOCOTYL 5 (HY5) protein at serine23 (S23) and S57 residues, contributing to the cold-induced accumulation of HY5 protein. HY5 directly binds to the promoter regions and stimulates the transcription of flavonoid biosynthesis genes. Further genetic analysis showed that CPK27 acts upstream of HY5, and the flavonoid biosynthesis pathway activated by CPK27 is HY5-dependent. Our study elucidates the regulatory mechanism whereby the CPK27-HY5 molecule integrates cold-triggered Ca2+ signals with flavonoid biosynthesis pathways to confer cold stress tolerance, thereby uncovering the key strategy for cold signal transduction.

Sea urchins have become significant mariculture species globally, and also serve as invertebrate model organisms in developmental biology. Cis-regulatory elements (enhancers) control development and physiology by regulating gene expression. Mutations that affect the function of these sequences may contribute to phenotypic diversity. Cis-regulatory targets offer new breeding potential for the future. Here, we use the CRISPR/Cas9 system to disrupt an enhancer of Endo16 in developing Lytechinus variegatus embryos, in consideration of the thorough research on Endo16’s regulatory region. We designed six gRNAs against Endo16 Module A (the most proximal region of regulatory sequences, which activates transcription in the vegetal plate and archenteron, specifically) and discovered that Endo16 Module A-disrupted embryos failed to undergo gastrulation at 20 h post fertilization. This result partly phenocopies morpholino knockdowns of Endo16. Moreover, we conducted qPCR and clone sequencing experiments to verify these results. Although mutations were not found regularly from sequencing affected individuals, we discuss some potential causes. In conclusion, our study provides a feasible and informative method for studying the function of cis-regulatory elements in sea urchins, and contributes to echinoderm precision breeding technology innovation and aquaculture industry development.

SummaryThe post‐translational modification of proteins enables cells to respond promptly to dynamic stimuli by controlling protein functions. In higher plants, SPINDLY (SPY) and SECRET AGENT (SEC) are two prominent O‐glycosylation enzymes that have both unique and overlapping roles; however, the effects of their O‐glycosylation on fruit ripening and the underlying mechanisms remain largely unknown. Here we report that SlSPY affects tomato fruit ripening. Using slspy mutants and two SlSPY‐OE lines, we provide biological evidence for the positive role of SlSPY in fruit ripening. We demonstrate that SlSPY regulates fruit ripening by changing the ethylene response in tomato. To further investigate the underlying mechanism, we identify a central regulator of ethylene signalling ETHYLENE INSENSITIVE 2 (EIN2) as a SlSPY interacting protein. SlSPY promotes the stability and nuclear accumulation of SlEIN2. Mass spectrometry analysis further identified that SlEIN2 has two potential sites Ser771 and Thr821 of O‐glycans modifications. Further study shows that SlEIN2 is essential for SlSPY in regulating fruit ripening in tomatoes. Collectively, our findings reveal a novel regulatory function of SlSPY in fruit and provide novel insights into the role of the SlSPY‐SlEIN2 module in tomato fruit ripening.

B-box (BBX) proteins are an important class of zinc finger transcription factors that play a critical role in plant growth and stress response. However, the mechanisms of how BBX proteins participate in the cold response in tomato remain unclear. Here, using approaches of reverse genetics, biochemical and molecular biology we characterized a BBX transcription factor, SlBBX17, which positively regulates cold tolerance in tomato (Solanum lycopersicum). Overexpressing SlBBX17 enhanced C-repeat binding factor (CBF)-dependent cold tolerance in tomato plants, whereas silencing SlBBX17 increased plant susceptibility to cold stress. Crucially, the positive role of SlBBX17 in CBF-dependent cold tolerance was dependent on ELONGATED HYPOCOTYL5 (HY5). SlBBX17 physically interacted with SlHY5 to directly promote the protein stability of SlHY5 and subsequently increased the transcriptional activity of SlHY5 on SlCBF genes under cold stress. Further experiments showed that cold-activated mitogen-activated protein kinases, SlMPK1 and SlMPK2, also physically interact with and phosphorylate SlBBX17 to enhance the interaction between SlBBX17 and SlHY5, leading to enhanced CBF-dependent cold tolerance. Collectively, the study unveiled a mechanistic framework by which SlMPK1/2-SlBBX17-SlHY5 regulated transcription of SlCBFs to enhance cold tolerance, thereby shedding light on the molecular mechanisms of how plants respond to cold stress via multiple transcription factors.

The sea cucumber Apostichopus japonicus has important nutritional and medicinal value. Unfortunately, we know little of the source of active chemicals in this animal, but the plentiful pigments of these animals are thought to function in intriguing ways for translation into clinical and food chemistry usage. Here, we found key cell groups with the gene activity predicted for the color morphology of sea cucumber body using single-cell RNA-seq. We refer to these cell populations as melanocytes and quinocytes, which are responsible for the synthesis of melanin and quinone pigments, respectively. We integrated analysis of pigment biochemistry with the transcript profiles to illuminate the molecular mechanisms regulating distinct pigment formation in echinoderms. In concert with the correlated pigment analysis from each color morph, this study expands our understanding of medically important pigment production, as well as the genetic mechanisms for color morphs, and provides deep datasets for exploring advancements in the fields of bioactives and nutraceuticals.

Chilling temperatures induce an increase in cytoplasmic calcium (Ca2+) ions to transmit cold signals, but the precise role of Calmodulins (CaMs), a type of Ca2+ sensor, in plant tolerance to cold stress remains elusive. In this study, we characterized a tomato (Solanum lycopersicum) CaM gene, CALMODULIN6 (CaM6), which responds to cold stimulus. Overexpressing CaM6 increased tomato sensitivity to cold stress whereas silencing CaM6 resulted in a cold-insensitive phenotype. We showed that CaM6 interacts with Inducer of CBF expression 1 (ICE1) in a Ca2+-independent process and ICE1 contributes to cold tolerance in tomato plants. By integrating RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) assays, we revealed that ICE1 directly altered the expression of 76 downstream cold-responsive (COR) genes that potentially confer cold tolerance to tomato plants. Moreover, the physical interaction of CaM6 with ICE1 attenuated ICE1 transcriptional activity during cold stress. These findings reveal that CaM6 attenuates the cold tolerance of tomato plants by suppressing ICE1-dependent COR gene expression. We propose a CaM6/ICE1 module in which ICE1 is epistatic to CaM6 under cold stress. Our study sheds light on the mechanism of plant response to cold stress and reveals CaM6 is involved in the regulation of ICE1.

\u3010Objective\u3011<\/strong>This study analyzed the effects of supplementary far-red light (FR) on the growth and abiotic stress tolerance of pepper seedlings, aiming to provide a theoretical basis on precise light environments for cultivating high quality vegetable seedlings.<\/p><\/sec>\u3010Method\u3011<\/strong>In this study, Bola Hongshuai pepper cult

[目的]研究补照适量远红光(FR)对辣椒幼苗生长发育和非生物胁迫抗性的调控作用,旨在为实际生产过程中利用精确的光环境调控手段培育壮苗提供理论依据.[方法]以辣椒'博辣红帅'品种为研究材料,将苗龄7 d的辣椒幼苗置于LED光源对照光谱(NL;红R/蓝B=3/1,光量子通量密度PPFD为150 μmol·m-2·s-1)及在此基础上分别补充10 μmol.m-2·s-1远红光(6％FR)、20 μmol·m-2·s-1远红光(13％FR)和30 μmol·m-2·s-1远红光(20％FR)的处理组光谱环境条件下培养,并于苗龄21 d时进行低温和干旱处理.通过测定生物量、抗性相关基因表达、抗氧化酶活性、激素含量、叶绿素荧光参数以及叶片相对电导率等,探究补充6％FR对辣椒幼苗生长和非生物胁迫抗性的影响.[结果]与对照光谱相比,补充6％FR显著提高了辣椒幼苗株高、茎粗、干鲜重以及壮苗指数(P＜0.05).低温胁迫下,相比于对照光谱组,补充6％FR显著提高了辣椒叶片冷响应基因CBF1和抗氧化酶相关基因Cu/Zn-SOD、GR、APX、CAT,DHAR的表达水平,超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)活性比对照分别增加25.2％、53.6％、55.8％、72.7％和33.4％,抗逆相关激素脱落酸(ABA)的含量提高69.5％.同时,低温胁迫下补充6％FR后辣椒叶片PSII最大光化学效率(Fv/Fm)较对照光谱组显著升高,而相对电导率(REL)显著降低,表明补充6％FR缓解了低温下PSII光抑制和叶片细胞的损伤,提高了辣椒幼苗的耐冷性.此外,干旱胁迫下,相比于对照光谱组,补充6％FR使辣椒幼苗的抗氧化酶SOD、GR、APX、CAT、DHAR活性分别增加13.7％、38.0％.37.2％、27.6％和23.7％,ABA含量和PSII实际光化学效率(ΦPSⅡ)也显著升高,而REL则明显降低,表明补充6％FR减轻了干旱胁迫引起的PSII光抑制和膜脂过氧化,提高了辣椒幼苗的耐旱性.[结论]补充6％FR不仅可促使辣椒壮苗的形成,还可通过增加抗氧化酶活性和ABA含量提高辣椒幼苗对低温胁迫和干旱胁迫的抗性.

The closely related species Helicoverpa armigera (H. armigera) and Helicoverpa assulta (H. assulta) have different host plant ranges and share two principal components of sex pheromones but with reversed ratios. The antennae are the main olfactory organ of insects and play a crucial role in host plant selection and mate seeking. However, the genetic basis for gene expression divergence in the antennae of the two species is unclear. We performed an allele-specific expression (ASE) analysis in the antennal transcriptomes of the two species and their F1 hybrids, examining the connection between gene expression divergence and phenotypic differences. The results show that the proportion of genes classified as all cis was higher than that of all trans in males and reversed in females. The contribution of regulatory patterns to gene expression divergence in males was less than that in females, which explained the functional differentiation of male and female antennae. Among the five groups of F1 hybrids, the fertile males from the cross of H. armigera female and H. assulta male had the lowest proportion of misexpressed genes, and the inferred regulatory patterns were more accurate. By using this group of F1 hybrids, we discovered that cis-related regulations play a crucial role in gene expression divergence of sex pheromone perception-related proteins. These results are helpful for understanding how specific changes in the gene expression of olfactory-related genes can contribute to rapid evolutionary changes in important olfactory traits in closely related moths.

Photosynthetic pigments in higher plants, including chlorophyll and carotenoid, are crucial for photosynthesis and photoprotection. Previous studies have shown that nitric oxide (NO) plays a dual role in plant photosynthesis. However, how pigment biosynthesis is suppressed by NO remains unclear. In this study, we generated NO-accumulated gsnor mutants, applied exogenous NO donors, and used a series of methods, including reverse transcription quantitative PCR, immunoblotting, chromatin immunoprecipitation, electrophoretic mobility shift, dual-luciferase, and NO content assays, to explore the regulation of photosynthetic pigment biosynthesis by NO in tomato. We established that both endogenous and exogenous NO inhibited pigment accumulation and photosynthetic capacities. High levels of NO stimulated the degradation of LONG HYPOCOTYL 5 (HY5) protein and further inactivated the transcription of genes encoding protochlorophyllide oxidoreductase C (PORC) and phytoene synthase 2 (PSY2)—two enzymes that catalyze the rate-limiting steps in chlorophyll and carotenoid biosynthesis. Our findings provide a new insight into the mechanism of NO signaling in modulating HY5-mediated photosynthetic pigment biosynthesis at the transcriptional level in tomato plants.

Changes in developmental gene regulatory networks (dGRNs) underlie much of the diversity of life, but the evolutionary mechanisms that operate on regulatory interactions remain poorly understood. Closely related species with extreme phenotypic divergence provide a valuable window into the genetic and molecular basis for changes in dGRNs and their relationship to adaptive changes in organismal traits. Here we analyse genomes, epigenomes and transcriptomes during early development in two Heliocidaris sea urchin species that exhibit highly divergent life histories and in an outgroup species. Positive selection and chromatin accessibility modifications within putative regulatory elements are enriched on the branch leading to the derived life history, particularly near dGRN genes. Single-cell transcriptomes reveal a dramatic delay in cell fate specification in the derived state, which also has far fewer open chromatin regions, especially near conserved cell fate specification genes. Experimentally perturbing key transcription factors reveals profound evolutionary changes to early embryonic patterning events, disrupting regulatory interactions previously conserved for ~225 million years. These results demonstrate that natural selection can rapidly reshape developmental gene expression on a broad scale when selective regimes abruptly change. More broadly, even highly conserved dGRNs and patterning mechanisms in the early embryo remain evolvable under appropriate ecological circumstances.

Activation of Wnt/β-catenin (cWnt) signaling at the future posterior end of early bilaterian embryos is a highly conserved mechanism for establishing the anterior-posterior (AP) axis. Moreover, inhibition of cWnt at the anterior end is required for development of anterior structures in many deuterostome taxa. This phenomenon, which occurs around the time of gastrulation, has been fairly well characterized, but the significance of intracellular inhibition of cWnt signaling in cleavage-stage deuterostome embryos for normal AP patterning is less well understood. To investigate this process in an invertebrate deuterostome, we defined Axin function in early sea urchin embryos. Axin is ubiquitously expressed at relatively high levels in early embryos and functional analysis revealed that Axin suppresses posterior cell fates in anterior blastomeres by blocking ectopic cWnt activation in these cells. Structure-function analysis of sea urchin Axin demonstrated that only its GSK-3β-binding domain is required for cWnt inhibition. These observations and results in other deuterostomes suggest that Axin plays a crucial conserved role in embryonic AP patterning by preventing cWnt activation in multipotent early blastomeres, thus protecting them from assuming ectopic cell fates.

An epithelial-mesenchymal transition (EMT) occurs in almost every metazoan embryo at the time mesoderm begins to differentiate. Several embryos have a long record as models for studying an EMT given that a known population of cells enters the EMT at a known time thereby enabling a detailed study of the process. Often, however, it is difficult to learn the molecular details of these model EMT systems because the transitioning cells are a minority of the population of cells in the embryo and in most cases there is an inability to isolate that population. Here we provide a method that enables an examination of genes expressed before, during, and after the EMT with a focus on just the cells that undergo the transition. Single cell RNA-seq (scRNA-seq) has advanced as a technology making it feasible to study the trajectory of gene expression specifically in the cells of interest, in vivo, and without the background noise of other cell populations. The sea urchin skeletogenic cells constitute only 5% of the total number of cells in the embryo yet with scRNA-seq it is possible to study the genes expressed by these cells without background noise. This approach, though not perfect, adds a new tool for uncovering the mechanism of EMT in this cell type.

The induction of C-repeat binding factors (CBFs) is crucial for plant survival at low temperatures. Therefore, understanding the mechanisms that regulate CBF transcription is vital for the future development of crops with increased cold tolerance. Here, we provide evidence for the existence of a LONG HYPOCOTYL 5 (HY5)-MYB15-CBFs transcriptional cascade that plays a crucial role in the cold response in tomato. The exposure of tomato plants to cold (4°C) increased the levels of HY5, MYB15 and CBFs transcripts. Moreover, mutations in HY5 or MYB15 decreased the levels of CBF transcripts. In contrast, overexpression of HY5 or MYB15 increased CBF transcript abundance. Crucially, the HY5 transcription factor activated the expression of MYB15 by directly binding to the promoter region, while both HY5 and MYB15 activated the expression of CBF1, CBF2 and CBF3. Taken together, these data show that HY5 can directly regulate CBF transcript levels, and also influence CBF expression indirectly via MYB15. The coordinated action of HY5 and MYB15 allows precise regulation of CBF expression and subsequent cold tolerance. These findings provide an improved understanding of the molecular mechanisms affording transcriptional regulation of CBFs, which can be exploited in the future to enhance cold tolerance in crops.

The ability to interpret daily and seasonal fluctuations, latitudinal and vegetation canopy variations in light and temperature signals is essential for plant survival. However, the precise molecular mechanisms transducing the signals from light and temperature perception to maintain plant growth and adaptation remain elusive. We show that far-red light induces PHYTOCHROME-INTERACTING TRANSCRIPTION 4 (SlPIF4) accumulation under low-temperature conditions via phytochrome A in Solanum lycopersicum (tomato). Reverse genetic approaches revealed that knocking out SlPIF4 increases cold susceptibility, while overexpressing SlPIF4 enhances cold tolerance in tomato plants. SlPIF4 not only directly binds to the promoters of the C-REPEAT BINDING FACTOR (SlCBF) genes and activates their expression but also regulates plant hormone biosynthesis and signals, including abscisic acid, jasmonate and gibberellin (GA), in response to low temperature. Moreover, SlPIF4 directly activates the SlDELLA gene (GA-INSENSITIVE 4, SlGAI4) under cold stress, and SlGAI4 positively regulates cold tolerance. Additionally, SlGAI4 represses accumulation of the SlPIF4 protein, thus forming multiple coherent feed-forward loops. Our results reveal that plants integrate light and temperature signals to better adapt to cold stress through shared hormone pathways and transcriptional regulators, which may provide a comprehensive understanding of plant growth and survival in a changing environment.

围绕干旱、盐渍、重金属和温度胁迫等非生物逆境,综述了丛枝菌根在提高园艺植物抗逆性中的作用,阐述了丛枝菌根特异性转录因子调控植物抗逆性的分子机制,探讨了植物生长调节剂、光环境、CO2浓度、营养元素等外源因子在增强丛枝菌根抗逆性效应上的作用,以期为利用丛枝菌根提高园艺植物抗逆性提供参考.