An omics strategy increasingly improves the discovery of genetic loci and genes for seed-coat color formation in soybean.

The phenotypic color of seeds is a complex agronomic trait and has economic and biological significance. The genetic control and molecular regulation mechanisms have been extensively studied. Here, we used a multi-omics strategy to explore the color formation in soybean seeds at a big data scale. We identified 13 large quantitative trait loci (QTL) for color with bulk segregating analysis in recombinant inbreeding lines. GWAS analysis of colors and decomposed attributes in 763 germplasms revealed associated SNP sites perfectly falling in five major QTL, suggesting inherited regulation on color during natural selection. Further transcriptomics analysis before and after color accumulation revealed 182 differentially expression genes (DEGs) in the five QTL, including known genes CHS, MYB, and F3'H involved in pigment accumulation. More DEGs with consistently upregulation or downregulation were identified as shared regulatory genes for two or more color formations while some DEGs were only for a specific color formation. For example, five upregulated DEGs in QTL qSC-3 were in flavonoid biosynthesis responsible for black and brown seed. The DEG (Glyma.08G085400) was identified in the purple seed only, which encodes gibberellin 2-beta-dioxygenase in the metabolism of colorful terpenoids. The candidate genes are involved in flavonoid biosynthesis, transcription factor regulation, gibberellin and terpenoid metabolism, photosynthesis, ascorbate and aldarate metabolism, and lipid metabolism. Seven differentially expressed transcription factors were also speculated that may regulate color formation, including a known MYB. The finds expand QTL and gene candidates for color formation, which could guide to breed better cultivars with designed colors. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01414-z.

QTL Mapping of Soybean (Glycine max) Vine Growth Habit Trait.

The vine growth habit (VGH) is a notable property of wild soybean plants that also holds a high degree of importance in domestication as it can preclude using these wild cultivars for breeding and improving domesticated soybeans. Here, a bulked segregant analysis (BSA) approach was employed to study the genetic etiology of the VGH in soybean plants by integrating linkage mapping and population sequencing approaches. To develop a recombinant inbred line (RIL) population, the cultivated Zhongdou41 (ZD41) soybean cultivar was bred with ZYD02787, a wild soybean accession. The VGH status of each line in the resultant population was assessed, ultimately leading to the identification of six and nine QTLs from the BSA sequencing of the F4 population and F6-F8 population sequence mapping, respectively. One QTL shared across these analyzed generations was detected on chromosome 19. Three other QTLs detected by BSA-seq were validated and localized to the 90.93 kb, 2.9 Mb, and 602.08 kb regions of chromosomes 6 and 13, harboring 14, 53, and 4 genes, respectively. Three consistent VGH-related QTLs located on chromosomes 2 and 19 were detected in a minimum of three environments, while an additional six loci on chromosomes 2, 10, 13, and 18 were detected in at least two environments via ICIM mapping. Of all the detected loci, five had been reported previously whereas seven represent novel QTLs. Together, these data offer new insights into the genetic basis of the VGH in soybean plants, providing a rational basis to inform the use of wild accessions in future breeding efforts.

Genome-Wide Identification of Binding Sites for SmTCP7a Transcription Factors of Eggplant during Bacterial Wilt Resistance by ChIP-Seq.

Teosinte branched 1/cycloidea/proliferating cell factor (TCP) transcription factors play a key role in the regulation of plant biotic and abiotic stresses. In this study, our results show that SmTCP7a positively regulated bacterial wilt that was caused by Ralstonia solanacearum. ChIP-seq was conducted to analyze the transcriptional regulation mechanism of SmTCP7a before (R0 h) and 48 h after infection (R48 h). SmTCP7a regulated a total of 92 and 91 peak-associated genes in R0 h and R48 h, respectively. A KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis showed that phenylpropanoid biosynthesis, MAPK (mitogen-activated protein kinas) signaling pathway, plant hormone signal transduction and plant-pathogen interactions were involved. The difference in peaks between R0 h and R48 h showed that there were three peak-associated genes that were modulated by infection. A better understanding of the potential target genes of SmTCP7a in response to R. solanacearum will provide a comprehensive understanding of the SmTCP7a regulatory mechanism during the eggplant defense response to bacterial wilt.

The SlTPL3-SlWUS module regulates multi-locule formation in tomato by modulating auxin and gibberellin levels in the shoot apical meristem.

Malformed fruits depreciate a plant's market value. In tomato (Solanum lycopersicum), fruit malformation is associated with the multi-locule trait, which involves genes regulating shoot apical meristem (SAM) development. The expression pattern of TOPLESS3 (SlTPL3) throughout SAM development prompted us to investigate its functional significance via RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing. Lower SlTPL3 transcript levels resulted in larger fruits with more locules and larger SAMs at the 5 d after germination (DAG5) stage. Differentially expressed genes in the SAM of wild-type (WT) and SlTPL3-RNAi plants, identified by transcriptome deep sequencing (RNA-seq), were enriched in the gibberellin (GA) biosynthesis and plant hormone signaling pathways. Moreover, exogenous auxin and paclobutrazol treatments rescued the multi-locule phenotype, indicating that SlTPL3 affects SAM size by mediating auxin and GA levels in the SAM. Furthermore, SlTPL3 interacted with WUSCHEL (SlWUS), which plays an important role in SAM size maintenance. We conducted RNA-seq and DNA affinity purification followed by sequencing (DAP-seq) analyses to identify the genes regulated by SlTPL3 and SlWUS in the SAM and to determine how they regulate SAM size. We detected 24 overlapping genes regulated by SlTPL3 and SlWUS and harboring an SlWUS-binding motif in their promoters. Furthermore, functional annotation revealed a notable enrichment for functions in auxin transport, auxin signal transduction, and GA biosynthesis. Dual-luciferase assays also revealed that SlTPL3 enhances SlWUS-mediated regulation (repression and activation) of SlPIN3 and SlGA2ox4 transcription, indicating that the SlTPL3-SlWUS module regulates SAM size by mediating auxin distribution and GA levels, and perturbations of this module result in enlarged SAM. These results provide novel insights into the molecular mechanism of SAM maintenance and locule formation in tomato and highlight the SlTPL3-SlWUS module as a key regulator.

Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit.

Auxin response factors (ARFs) play important roles in various plant physiological processes; however, knowledge of the exact role of ARFs in plant responses to water deficit is limited. In this study, SlARF4, a member of the ARF family, was functionally characterized under water deficit. Real-time fluorescence quantitative polymerase chain reaction (PCR) and ß-glucuronidase (GUS) staining showed that water deficit and abscisic acid (ABA) treatment reduced the expression of SlARF4. SlARF4 was expressed in the vascular bundles and guard cells of tomato stomata. Loss of function of SlARF4 (arf4) by using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 (CRISPR/Cas 9) technology enhanced plant resistance to water stress and rehydration ability. The arf4 mutant plants exhibited curly leaves and a thick stem. Malondialdehyde content was significantly lower in arf4 mutants than in wildtype plants under water stress; furthermore, arf4 mutants showed higher content of antioxidant substances, superoxide dismutase, actual photochemical efficiency of photosystem II (PSII), and catalase activities. Stomatal and vascular bundle morphology was changed in arf4 mutants. We identified 628 differentially expressed genes specifically expressed under water deficit in arf4 mutants; six of these genes, including ABA signaling pathway-related genes, were differentially expressed between the wildtype and arf4 mutants under water deficit and unlimited water supply. Auxin responsive element (AuxRE) elements were found in these genes' promoters indicating that SlARF4 participates in ABA signaling pathways by regulating the expression of SlABI5/ABF and SCL3, thereby influencing stomatal morphology and vascular bundle development and ultimately improving plant resistance to water deficit.

The SlHB8 Acts as a Negative Regulator in Stem Development and Lignin Biosynthesis.

The stem is an important organ in supporting plant body, transporting nutrients and communicating signals for plant growing. However, studies on the regulation of stem development in tomato are rather limited. In our study, we demonstrated that SlHB8 negatively regulated tomato stem development. SlHB8 belongs to homeo domain-leucine zipper Class III gene family transcription factors and expressed in all the organs examined including root, stem, leaves, flower, and fruit. Among these tissues, SlHB8 showed stable high expression level during tomato stem development. Overexpression of SlHB8 gene decreased stem diameter with inhibited xylem width and xylem cell layers, while loss of function of SlHB8gene increased the stem diameter and xylem width. The contents of lignin were decreased both in leaves and stems of SlHB8 overexpression plants. RNA-seq analysis on the stems of wild type and SlHB8 transgenic plants showed that the 116 DEGs (differential expressed genes) with reversible expression profiles in SlHB8-ox and SlHB8-cr plants were significantly enriched in the phenylpropanoid biosynthesis pathway and plant-pathogen pathway which were related to lignin biosynthesis and disease resistance. Meanwhile, the key genes involved in the lignin biosynthesis pathway such as SlCCR (cinnamoyl-CoA reductase), SlCYP73A14/C4H (cinnamate 4-hydroxylase), SlC3H (coumarate 3-hydroxylase) and SlCAD (cinnamoyl alcohol dehydrogenase) were down-regulated in both stem and leaves of SlHB8 overexpression plants, indicating a negative regulatory role of SlHB8 in the lignin biosynthesis and stem development.

The SlARF4-SlHB8 regulatory module mediates leaf rolling in tomato.

Leaf is the main photosynthetic organ in plants and the primary energy source all along the plant life. Given the beneficial role of leaf rolling in improving photosynthetic efficiency and yield in specific environmental conditions, a better understanding of the factors and molecular mechanisms underlying this process is highly suited. Previously, the SlARF4 knocking out mutant exhibited upward curly leaf showed higher resistance to water deficit which driving us to uncover the function of SlARF4 in regulating the curly leaf formation. In this study, we unraveled the unexplored role of the SlARF4-SlHB8 module of transcription factors in the development of leaf rolling. Both SlARF4 loss-of-function and SlHB8 overexpressing tomato plants exhibited upward-rolled leaves, reflecting the active role of the two genes in controlling leaf rolling. Dual-luciferase reporter assays and phenotypic analysis of hybrid progenies suggested that SlHB8 acts downstream of SlARF4 in curly leaf formation. SlARF4 and SlHB8 influence the development of leaf palisade tissues via modulating the expression of genes associated with curly leaf formation. SEM analysis revealed no significant differences in leaf epidermal cells between the two leaf-rolling mutants and the wild type, indicating that curly leaves of arf4 and SlHB8-OE do not result from the asymmetric leaf epidermal cell growth. Our data provide novel insight into the molecular mechanism of abaxial-adaxial determination involving SlARF4 and SlHB8 and reveals that leaf rolling operates via different regulation mechanisms in tomato and Arabidopsis model plant.

The SlHB8 acts as a negative regulator in tapetum development and pollen wall formation in Tomato.

Pollen development is crucial for the fruit setting process of tomatoes, but the underlying regulatory mechanism remains to be elucidated. Here, we report the isolation of one HD-Zip III family transcription factor, SlHB8, whose expression levels decreased as pollen development progressed. SlHB8 knockout using CRISPR/Cas9 increased pollen activity, subsequently inducing fruit setting, whereas overexpression displayed opposite phenotypes. Overexpression lines under control of the 35 s and p2A11 promoters revealed that SlHB8 reduced pollen activity by affecting early pollen development. Transmission electron microscopy and TUNEL analyses showed that SlHB8 accelerated tapetum degradation, leading to collapsed and infertile pollen without an intine and an abnormal exine. RNA-seq analysis of tomato anthers at the tetrad stage showed that SlHB8 positively regulates SPL/NZZ expression and the tapetum programmed cell death conserved genetic pathway DYT1-TDF1-AMS-MYB80 as well as other genes related to tapetum and pollen wall development. In addition, DNA affinity purification sequencing, electrophoretic mobility shift assay, yeast one-hybrid assay and dual-luciferase assay revealed SlHB8 directly activated the expression of genes related to pollen wall development. The study findings demonstrate that SlHB8 is involved in tapetum development and degradation and plays an important role in anther development.

SlTPL1 Silencing Induces Facultative Parthenocarpy in Tomato.

Facultative parthenocarpy is of great practical value. However, the molecular mechanism underlying facultative parthenocarpy remains elusive. Transcriptional co-repressors (TPL) act as a central regulatory hub controlling all nine phytohormone pathways. Previously, we proved that SlTPLs participate in the auxin signaling pathway by interacting with auxin/indole acetic acid (Aux/IAAs) in tomato; however, their function in fruit development has not been studied. In addition to their high expression levels during flower development, the interaction between SlTPL1 and SlIAA9 stimulated the investigation of its functional significance via RNA interference (RNAi) technology, whereby the translation of a protein is prevented by selective degradation of its encoded mRNA. Down-regulation of SlTPL1 resulted in facultative parthenocarpy. Plants of SlTPL1-RNAi transgenic lines produced similar fruits which did not show any pleiotropic effects under normal conditions. However, they produced seedless fruits upon emasculation and under heat stress conditions. Furthermore, SlTPL1-RNAi flower buds contained higher levels of cytokinins and lower levels of abscisic acid. To reveal how SlTPL1 regulates facultative parthenocarpy, RNA-seq was performed to identify genes regulated by SlTPL1 in ovaries before and after fruit set. The results showed that down-regulation of SlTPL1 resulted in reduced expression levels of cytokinin metabolism-related genes, and all transcription factors such as MYB, CDF, and ERFs. Conversely, down-regulation of SlTPL1 induced the expression of genes related to cell wall and cytoskeleton organization. These data provide novel insights into the molecular mechanism of facultative tomato parthenocarpy and identify SlTPL1 as a key factor regulating these processes.

Effect of bevacizumab on expression level of GLI1 and ING4 in colon cancer animal model.

This study aimed to investigate the effect of bevacizumab on GLI1 and ING4 expression in colon cancer animal model. Colon cancer model in rats was induced by azoxymethane (AOM). Bevacizumab was used for the treatment of colon cancer rats. Tumor volume and weight were measured, tumor growth curve was visualized and tumor inhibition rate was calculated. GLI1 and ING4 of colon cancer cells were silencing expressed. Western blot analysis was used to detect the expressions of GLI1, ING4, caspase-3, Bax, ß-catenin, Bcl2, PTEN, PI3K, Akt, NF-κB. The apoptosis rate was detected by flow cytometry. MTT assay was used to detect cell activity to get IC50 value. After AOM induced colon cancer model in rats, the expressions of ING4, caspase-3, Bax and PTEN were downregulated, the expressions of GLI1, ß-catenin, Bcl2, PI3K, Akt and NF-κB were upregulated and the apoptosis rate was downregulated. After bevacizumab treatment, the tumor volume and weight decreased, the expressions of ING4, caspase-3, Bax, PTEN were upregulated, the expressions of GLI1, ß-catenin, Bcl2, PI3K, Akt, NF-κB were downregulated, and the cell apoptosis rate increased. Cell experiments showed that GLI1 promotes tumor growth and reduces the sensitivity of bevacizumab, while ING4 inhibits tumor growth and increases the sensitivity of bevacizumab. Bevacizumab inhibits the growth of colon cancer tumor by upregulating ING4 and downregulating GLI1.