An optimum study on the laser scanning confocal microscopy techniques for BiFC assay using plant protoplast.

BACKGROUND: The bimolecular fluorescence complementation (BiFC) assay is commonly used for investigating protein-protein interactions. While several BiFC detection systems have been developed, there is a limited amount of research focused on using laser scanning confocal microscope (LSCM) techniques to observe protoplasts. Protoplasts are more susceptible to damage and instability compared to their original cell state due to the preparation treatments they undergo, which makes it challenging for researchers to manipulate them during observation under LSCMs. Therefore, it is crucial to utilize microscope techniques properly and efficiently in BiFC assays. RESULTS: When the target fluorescence is weak, the autofluorescence of chloroplast particles in protoplasts can interfere with the detection of BiFC signals localized in the nuclear region. Spectrum analysis revealed that chloroplast autofluorescence can be excited by lasers of various types, with the highest fluorescence signal observed at around 660 nm. Furthermore, our investigation into the impact of different pipette tips on the integrity of protoplast samples indicated that the utilization of cut tips with larger openings can mitigate cell breakage. We presented a workflow of LSCM techniques for investigating protoplast BiFC and discussed the microscopic manipulation involved in sample preparation and image capturing. CONCLUSION: When the BiFC signals are weak, they may be affected by chloroplast autofluorescence. However, when used properly, the autofluorescence of chloroplasts can serve as an excellent internal marker for effectively distinguishing other signals. In combination with other findings, this study can provide valuable reference for researchers conducting BiFC assays and related studies.

B. subtilis CNBG-PGPR-1 induces methionine to regulate ethylene pathway and ROS scavenging for improving salt tolerance of tomato.

Soil salinity severely threatens plant growth and crop yields. The utilization of PGPR is an effective strategy for enhancing plant salt tolerance, but the mechanisms involved in this process have rarely been reported. In this study, we investigated the effects of Bacillus subtilis CNBG-PGPR-1 on improving plant salt tolerance and elucidated the molecular pathways involved. The results showed that CNBG-PGPR-1 significantly improved the cellular homeostasis and photosynthetic efficiency of leaves and reduced ion toxicity and osmotic stress caused by salt in tomato. Transcriptome analysis uncovered that CNBG-PGPR-1 enhanced plant salt tolerance through the activation of complex molecular pathways, with plant hormone signal transduction playing an important role. Comparative analysis and pharmacological experiments confirmed that the ethylene pathway was closely related to the beneficial effect of CNBG-PGPR-1 on improving plant salt tolerance. Furthermore, we found that methionine, a precursor of ethylene synthesis, significantly accumulated in response to CNBG-PGPR-1 in tomato. Exogenous L-methionine largely mimicked the beneficial effects of CNBG-PGPR-1 and activated the expression of ethylene pathway-related genes, indicating CNBG-PGPR-1 induces methionine accumulation to regulate the ethylene pathway in tomato. Finally, CNBG-PGPR-1 reduced salt-induced ROS by activating ROS scavenger-encoding genes, mainly involved in GSH metabolism and POD-related genes, which were also closely linked to methionine metabolism. Overall, our studies demonstrate that CNBG-PGPR-1-induced methionine is a key regulator in enhancing plant salt tolerance through the ethylene pathway and ROS scavenging, providing a novel understanding of the mechanism by which beneficial microbes improve plant salt tolerance.

Transcriptome Analysis Revealed that GhPP2C43-A Negatively Regulates Salinity Tolerance in an Introgression Line from a Semi-Wild Upland Cotton.

Salt damage is a major threat to sustainable cotton production owing to the limited arable land in China, which is mainly occupied by the production of staple food crops. Salt-stress-tolerant cotton varieties are lacking in production, and the mechanisms underpinning salt stress tolerance in cotton remain enigmatic. Here, DM37, an intraspecific introgression line from Gossypium hirsutum race yucatanense acc TX-1046 into the G. hirsutum acc TM-1 background, was found to be highly tolerant to salt stress. Its seed germination rate and germination potential were significantly higher than those of the recipient TM-1 under salt stress. Physiological analysis showed that DM37 had a higher proline content and peroxidase activity and lower Na+/K+ ratios at the seedling stage, which is consistent with a higher seedling survival rate after durable salt stress. Furthermore, comparative transcriptome analysis revealed that responsive patterns to salt stress in DM37 were different from those in TM-1. Weighted correlation network analysis demonstrated that co-expression modules associated with salt stress in DM37 also differed from those in TM-1. From this analysis, GhPP2C43-A, a phosphatase gene, was found to exhibit negative regulation of salt stress tolerance verified by virus-induced gene silencing and the genration of transgenic Arabidopsis. Gene expression showed that GhPP2C43-A in TM-1 was induced by durable salt stress but not in DM37, probably attributable to a variation in the cis-element in its promoter, thereby conferring different salt stress tolerance. These results provide new genes/germplasms from semi-wild cotton in salt-stress-tolerant cotton breeding, as well as new insight into the mechanisms underpinning salt stress tolerance in cotton.

TIP41L, a putative candidate gene conferring both seed size and boll weight, was fine-mapped in an introgression line of Gossypium hirsutum-Gossypium arboreum.

QTLs for yield-related traits in tetraploid cotton have been widely mapped, but QTLs introduced from diploid species into tetraploid cotton background remain uninvolved. Here, a stable introgression line with the traits of small boll and seed on Chr. A12, IL197 derived from Gossypium hirsutum (2n = AADD = 52) × Gossypium arboreum (2n = AA = 26), was employed to construct the F2 and F3 secondary populations for fine-mapping QTLs of yield-related traits. QTL analysis showed eight QTLs were detected for three traits, boll weight (BW), seed index (SI, one-hundred-seed weight in g), and lint percentage, with 3.94-28.13 % of the phenotypic variance explained. Of them, a stable major QTL, q(BW + SI)-A12-1 controlling both BW and SI and covering the shortest region in Chr. A12, was further narrowed into a 60.09 kb-interval through substitution mapping. Finally, five candidate genes were detected in the interval. The qRT-PCR analysis revealed only TIP41-like family protein (TIP41L) kept up-regulated expression and significantly lower in TM-1 than that in IL197 from -1 DPA to 15 DPA during cotton boll rapid developmental stage. Therefore, TIP41L gene is speculated as the most likely candidate gene. Comparative analysis with the other four allotetraploid species showed TIP41L gene was probably diverged after the formation of allotetraploid cotton, which may be selected and swept during domestication of modern upland cotton because small boll and seed are detrimental to fibre yield of cotton. This research would lay a solid foundation for further elucidating the molecular mechanism of cotton boll and seed development.

Genome-Wide Introgression and Quantitative Trait Locus Mapping Reveals the Potential of Asian Cotton (Gossypium arboreum) in Improving Upland Cotton (Gossypium hirsutum).

Gossypium arboreum (2n=2x=26, A2), the putative progenitor of the At-subgenome of Gossypium hirsutum (2n=4x=52, AD), is a repository of genes of interesting that have been eliminated during evolution/domestication of G. hirsutum. However, its valuable genes remain untapped so far due to species isolation. Here, using a synthetic amphiploid (AADDA2A2) previously reported, we developed a set of 289 G. arboreum chromosome segment introgression lines (ILs) in G. hirsutum by expanding the backcrossing population and through precise marker-assisted selection (MAS) although complex chromosomal structural variations existed between parents which severely hindered introgression. Our results showed the total coverage length of introgressed segments was 1,116.29 Mb, representing 78.48% of the At-subgenome in the G. hirsutum background, with an average segment-length of 8.69 Mb. A total of 81 co- quantitative trait loci (QTLs) for yield and fiber quality were identified by both the RSTEP-ADD-based QTL mapping and the genome-wide association study (GWAS) analysis, with 1.01-24.78% of the phenotypic variance explained. Most QTLs for boll traits showed negative additive effects, but G. arboreum still has the potential to improve boll-number traits in G. hirsutum. Most QTLs for fiber quality showed negative additive effects, implying these QTLs were domesticated in G. hirsutum compared with G. arboreum and, a small quantity of fiber quality QTLs showing positive additive effects, conversely; however, indicates that G. arboreum has the underlying genes of enhancing fiber quality of G. hirsutum. This study provides new insights into the breeding genetic potential of G. arboreum, lays the foundation for further mining favorable genes of interest, and provides guidance for inter-ploidy gene transference from relatives into cultivated crops.

Fine-mapping and candidate gene analysis of qFS-Chr. D02, a QTL for fibre strength introgressed from a semi-wild cotton into Gossypium hirsutum.

Fibre strength (FS) is an important quality attribute in the modern textile industry, which is genetically controlled by quantitative trait loci (QTLs). Fine-mapping stable QTLs for FS to identify candidate genes would be valuable for uncovering the genetic basis of fibre quality traits in cotton. Here, a single segment introgression line, IL-D2-2, from the cross of (TM-1×TX-1046) reported in our previous studies, was found to have significantly improved FS compared with the recurrent parent TM-1. To fine-map the QTLs of the FS, we further crossed IL-D2-2 with its recurrent parent TM-1 to produce F2 and F2:3 populations. QTL analysis and substitution mapping showed qFS-Chr. D02 was anchored into a 550.66 kb-interval between two markers, INTR1027 and JESPR-231. This interval contained 67 genes, among which 27 genes related to cell-wall synthesis were selected to conduct qRT-PCR. The results revealed seven genes were expressed significantly differently during the fibre secondary-wall-thickening stage (10-25 days post-anthesis), three being upregulated and four downregulated in IL-D2-2. Both GH_D02G2269 (UDP-glucosyl transferase 84B1) and GH_D02G2289 (unknown function (DUF869)) with nonsynonymous SNPs in IL-D2-2 had significantly downregulated expression, suggesting they were candidates for qFS-Chr. D02. This research provides information about marker-assisted selection for cotton fibre strength improvement.

ELFN1-AS1 accelerates the proliferation and migration of colorectal cancer via regulation of miR-4644/TRIM44 axis.

Faced with the increasing colorectal cancer (CRC) cases, the interrogation of pivotal molecules in CRC appears to be vitally important. Long non-coding RNAs (lncRNAs) are well-known regulators of gene expression at transcriptional, post-transcriptional or epigenetic level, among which the competing endogenous RNA (ceRNA) network is a common way that lncRNAs exert their properties. The current study aimed to provide a new insight into improving the outcomes of CRC patients. Our study detected that ELFN1-AS1 expression was elevated in CRC tissues and cells, and ELFN1-AS1 upregulation was correlated with poor prognosis of CRC sufferers. Besides, it was viewed that ELFN1-AS1 knockdown impeded the proliferation and migration abilities as well as activated the apoptosis ability of CRC cells. In subsequence, mechanism assays also displayed that ELFN1-AS1 targeted miR-4644 to augment TRIM44 level. Finally, rescue experiments confirmed that TRIM44 took part in the ELFN1-AS1-medatied promotional influences on CRC cells proliferation and migration. In conclusion, ELFN1-AS1 exerted pro-proliferation, anti-apoptosis and pro-migration functions on CRC cells by acting as a sponge of miR-4644 to increase TRIM44 expression at mRNA and protein level, providing an additional molecule responsible for the carcinogenesis and progression for CRC.

QTL analysis for yield and fibre quality traits using three sets of introgression lines developed from three Gossypium hirsutum race stocks.

Upland cotton (Gossypium hirsutum L.) race stocks may possess desirable traits for the genetic improvement of cotton. Quantitative trait locus (QTL) analysis can assist in uncovering new alleles from unadapted race stocks. In this study, three sets of chromosome segment introgression lines (ILs) were developed from three backcrosses (BC3) between three race stocks, G. hirsutum races latifolium accs. TX-34 and TX-48 and punctatum acc. TX-114, as donor parents and Texas Marker-1 (TM-1) as the recurrent parent. Based on a total of 452 polymorphic simple sequence repeat (SSR) markers in BC3F2 genotyping, 149, 150 and 184 ILs were obtained from TM-1 × TX-34, TM-1 × TX-48 and TM-1 × TX-114, respectively. The average introgressed chromosomal segment length was 12.7 cM, and the total genetic distance was 3268 cM covering approximately 73.4% of the Upland cotton genome. The BC3F2, BC3F2:3 and BC3F2:4 progeny, which produced the ILs, were evaluated for yield and fibre quality traits. A total of 128 QTLs were detected, each of which explained 1.6-13.0% of the phenotypic variation. Thirty-five common QTLs related to eight traits were detected. Six QTL clusters were found on five chromosomes. Thirty-eight QTLs were previously unreported, and they may be footprints of cotton domestication. Domestication or artificial selection by humans successfully eliminated most unfavourable QTLs (21/38); however, some favourable QTLs (17/38) are not present in modern cultivars, demonstrating the importance of race stocks for improving cotton cultivars. The 26 elite ILs developed could be used to improve the yield and fibre quality components simultaneously. These results provide information on desirable QTLs for cotton improvement.

Mapping of fiber quality QTLs reveals useful variation and footprints of cotton domestication using introgression lines.

Fiber quality improvement is a driving force for further cotton domestication and breeding. Here, QTLs for fiber quality were mapped in 115 introgression lines (ILs) first developed from two intraspecific populations of cultivated and feral cotton landraces. A total of 60 QTLs were found, which explained 2.03-16.85% of the phenotypic variance found in fiber quality traits. A total of 36 markers were associated with five fiber traits, 33 of which were found to be associated with QTLs in multiple environments. In addition, nine pairs of common QTLs were identified; namely, one pair of QTLs for fiber elongation, three pairs for fiber length, three pairs for fiber strength and two pairs for micronaire (qMICs). All common QTLs had additive effects in the same direction in both IL populations. We also found five QTL clusters, allowing cotton breeders to focus their efforts on regions of QTLs with the highest percentages of phenotypic variance. Our results also reveal footprints of domestication; for example, fourteen QTLs with positive effects were found to have remained in modern cultivars during domestication, and two negative qMICs that had never been reported before were found, suggesting that the qMICs regions may be eliminated during artificial selection.