Plant regeneration via somatic embryogenesis (SE) is a key step during genetic engineering. In the current study, integrated widely targeted metabolomics and RNA sequencing were performed to investigate the dynamic metabolic and transcriptional profiling of cotton SE. Our data revealed that a total of 581 metabolites were present in nonembryogenic staged calli (NEC), primary embryogenic calli (PEC), and initiation staged globular embryos (GE). Of the differentially accumulated metabolites (DAMs), nucleotides, and lipids were specifically accumulated during embryogenic differentiation, whereas flavones and hydroxycinnamoyl derivatives were accumulated during somatic embryo development. Additionally, metabolites related to purine metabolism were significantly enriched in PEC vs. NEC, whereas in GE vs. PEC, DAMs were remarkably associated with flavonoid biosynthesis. An association analysis of the metabolome and transcriptome data indicated that purine metabolism and flavonoid biosynthesis were co-mapped based on the Kyoto encyclopedia of genes and genomes (KEGG) database. Moreover, purine metabolism-related genes associated with signal recognition, transcription, stress, and lipid binding were significantly upregulated. Moreover, several classic somatic embryogenesis (SE) genes were highly correlated with their corresponding metabolites that were involved in purine metabolism and flavonoid biosynthesis. The current study identified a series of potential metabolites and corresponding genes responsible for SE transdifferentiation, which provides a valuable foundation for a deeper understanding of the regulatory mechanisms underlying cell totipotency at the molecular and biochemical levels.

The somatic embryogenesis (SE) process of plants, as one of the typical responses to abiotic stresses with hormone, occurs through the dynamic expression of different proteins that constitute a complex regulatory network in biological activities and promotes plant totipotency. Plant SE includes two critical stages: primary embryogenic calli redifferentiation and somatic embryos development initiation, which leads to totipotency. The isobaric labels tandem mass tags (TMT) large-scale and quantitative proteomics technique was used to identify the dynamic protein expression changes in nonembryogenic calli (NEC), primary embryogenic calli (PEC) and globular embryos (GEs) of cotton. A total of 9369 proteins (6730 quantified) were identified; 805, 295 and 1242 differentially accumulated proteins (DAPs) were identified in PEC versus NEC, GEs versus PEC and GEs versus NEC, respectively. Eight hundred and five differentially abundant proteins were identified, 309 of which were upregulated and 496 down regulated in PEC compared with NEC. Of the 295 DAPs identified between GEs and PEC, 174 and 121 proteins were up- and down regulated, respectively. Of 1242 differentially abundant proteins, 584 and 658 proteins were up- and down regulated, respectively, in GEs versus NEC. We have also complemented the authenticity and accuracy of the proteomic analysis. Systematic analysis indicated that peroxidase, photosynthesis, environment stresses response processes, nitrogen metabolism, phytohormone response/signal transduction, transcription/posttranscription and modification were involved in somatic embryogenesis. The results generated in this study demonstrate a proteomic molecular basis and provide a valuable foundation for further investigation of the roles of DAPs in the process of SE transdifferentiation during cotton totipotency.

As a notable illustration of totipotency and plant regeneration, somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway, the key step for genetic engineering. Investigations examining the totipotency process are of great fundamental and practical importance in crop biotechnology. However, high-frequency regeneration of cotton via SE has been limited due to genotype-dependent response. The molecular basis deciphering SE genotype recalcitrance remains largely unexplored in cotton. In the current study, to comprehensively investigate the dynamic transcriptional profiling and gene regulatory patterns involved in SE process, a genome-wide RNA sequencing analysis was performed in two cotton genotypes with distinct embryogenic abilities, the highly embryogenic genotype Yuzao 1 (YZ) and the recalcitrant genotype Lumian 1 (LM). Three typical developmental staged cultures of early SE—hypocotyls (HY), nonembryogenic calli (NEC) and primary embryogenic calli (PEC)—were selected to establish the transcriptional profiles. Our data revealed that a total of 62,562 transcripts were present amongst different developmental stages in the two genotypes. Of these, 18,394 and 26,514 differentially expressed genes (DEGs) were identified during callus dedifferentiation (NEC-VS-HY) and embryogenic transdifferentiation (PEC-VS-NEC), respectively in the recalcitrant genotype, 21,842 and 22,343 DEGs in the highly embryogenic genotype. Furthermore, DEGs were clustered into six expression patterns during cotton SE process in the two genotypes. Moreover, functional enrichment analysis revealed that DEGs were significantly enriched in fatty acid, tryptophan and pyruvate metabolism in the highly embryogenic genotype and in DNA conformation change otherwise in the recalcitrant genotype. In addition, critical SE-associated expressed transcription factors, as well as alternative splicing events, were notably and preferentially activated during embryogenic transdifferentiation in the highly embryogenic genotype compared with the recalcitrant genotype. Taken together, by systematically comparing two genotypes with distinct embryogenic abilities, the findings in our study revealed a comprehensive overview of the dynamic gene regulatory patterns and uncharacterized complex regulatory pathways during cotton SE genotype-dependent response. Our work provides insights into the molecular basis and important gene resources for understanding the underlying genotype recalcitrance during SE process and plant regeneration, thereby holding great promise for accelerating the application of biotechnology to cotton for improving its breeding efficiency.

Kiwifruit (Actinidia chinensis) roots instead of fruits are widely used as Chinese medicine, but the functional metabolites remain unclear. In this study, we conducted comparative metabolome analysis between root and fruit in kiwifruit. A total of 410 metabolites were identified in the fruit and root tissues, and of them, 135 metabolites were annotated according to the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway. Moreover, 54 differentially expressed metabolites (DEMs) were shared in root and fruit, with 17 DEMs involved in the flavonoid pathway. Of the 17 DEMs, three flavonols (kaempferol-3-rhamnoside, L-Epicatechin and trifolin) and one dihydrochalcone (phloretin) showed the highest differences in the content level, suggesting that flavonols and dihydrochalcones may act as functional components in kiwifruit root. Transcriptome analysis revealed that genes related to flavonols and dihydrochalcones were highly expressed in root. Moreover, two AP2 transcription factors (TFs), AcRAP2-4 and AcAP2-4, were highly expressed in root, while one bHLH TF AcbHLH62 showed extremely low expression in root. The expression profiles of these TFs were similar to those of the genes related to flavonols and dihydrochalcones, suggesting they are key candidate genes controlling the flavonoid accumulation in kiwifruit. Our results provided an insight into the functional metabolites and their regulatory mechanism in kiwifruit root.

Green fiber (GF) is a naturally colored fiber. A limited understanding of its color formation mechanism restricts the improvement of colored cotton quality. This experiment used upland cotton green fiber germplasm 1-4560 and genetic inbred line TM-1; the lipid profiles of green fibers at 30 (white stage) and 35 days post-anthesis (DPA) (early greening stage), as well as those of TM-1 at the same stages, were revealed. Among the 109 differential types of lipids (DTLs) unique to GF, the content of phosphatidylserine PS (16:0_18:3) was significantly different at 30 and 35 DPA. It is speculated that this lipid is crucial for the pigment accumulation and color formation process of green fibers. The 197 DTLs unique to TM-1 may be involved in white fiber (WF) development. Among the shared DTLs in GF35 vs. GF30 and WF35 vs. WF30, sulfoquinovosyldiacyl-glycerol SQDG (18:1_18:1) displays a significant difference in the content change between green fibers and white fibers, potentially affecting color formation through changes in content. The enriched metabolic pathways in both comparison groups are relatively conserved. In the most significantly enriched glycerophospholipid metabolic pathway, 1-acyl-sn-glycero-3-phosphocholine (C04230) only appears in white cotton. This indicates differences in the metabolic pathways between white and green fibers, potentially related to different mechanisms of color formation and fiber development. These findings provide a new theoretical basis for studying cotton fiber development and offer important insights into the specific mechanism of green fiber color formation.

Long noncoding RNAs are involved in diverse biological processes in plants. Our recent study has revealed that an ABA-induced long intergenic noncoding RNA, ARTA, regulates both ABA and drought responses by blocking the nuclear import of a transcription factor, MYB7, through interacting with an importin β-like protein, SAD2. Here, we show that unlike MYB7, ARTA fails to disrupt interactions of SAD2 with the other two R2R3-MYB subgroup 4 members, MYB4 and MYB32. Consequently, the nuclear localizations of MYB4 and MYB32 remain unchanged upon alteration of ARTA expression. Furthermore, ARTA and MYB7 form a self-reinforcing feedback loop during Arabidopsis responses to ABA: ABA treatment induces ARTA expression, which in turn inhibits nuclear accumulation of MYB7, thereby deteriorating MYB7-mediated repression of ARTA and promoting ARTA production. This self-reinforcing feedback regulation elegantly integrates protein relocalization with transcriptional augmentation in the ABA response process, and provides a tunable molecular circuit for plant stress adaptation.