Genetic dissection of branch architecture in oilseed rape (Brassica napus L.) germplasm.

Branch architecture is an important factor influencing rapeseed planting density, mechanized harvest, and yield. However, its related genes and regulatory mechanisms remain largely unknown. In this study, branch angle (BA) and branch dispersion degree (BD) were used to evaluate branch architecture. Branch angle exhibited a dynamic change from an increase in the early stage to a gradual decrease until reaching a stable state. Cytological analysis showed that BA variation was mainly due to xylem size differences in the vascular bundle of the branch junction. The phenotypic analysis of 327 natural accessions revealed that BA in six environments ranged from 24.3° to 67.9°, and that BD in three environments varied from 4.20 cm to 21.4 cm, respectively. A total of 115 significant loci were detected through association mapping in three models (MLM, mrMLM, and FarmCPU), which explained 0.53%-19.4% of the phenotypic variations. Of them, 10 loci were repeatedly detected in different environments and models, one of which qBAD.A03-2 was verified as a stable QTL using a secondary segregation population. Totally, 1066 differentially expressed genes (DEGs) were identified between branch adaxial- and abaxial- sides from four extremely large or small BA/BD accessions through RNA sequencing. These DEGs were significantly enriched in the pathways related to auxin biosynthesis and transport as well as cell extension such as indole alkaloid biosynthesis, other glycan degradation, and fatty acid elongation. Four known candidate genes BnaA02g16500D (PIN1), BnaA03g10430D (PIN2), BnaC03g06250D (LAZY1), and BnaC06g20640D (ARF17) were identified by both GWAS and RNA-seq, all of which were involved in regulating the asymmetric distribution of auxins. Our identified association loci and candidate genes provide a theoretical basis for further study of gene cloning and genetic improvement of branch architecture.

GhTCE1-GhTCEE1 dimers regulate transcriptional reprogramming during wound-induced callus formation in cotton.

Wounded plant cells can form callus to seal the wound site. Alternatively, wounding can cause adventitious organogenesis or somatic embryogenesis. These distinct developmental pathways require specific cell fate decisions. Here, we identify GhTCE1, a basic helix-loop-helix family transcription factor, and its interacting partners as a central regulatory module of early cell fate transition during in vitro dedifferentiation of cotton (Gossypium hirsutum). RNAi- or CRISPR/Cas9-mediated loss of GhTCE1 function resulted in excessive accumulation of reactive oxygen species (ROS), arrested callus cell elongation, and increased adventitious organogenesis. In contrast, GhTCE1-overexpressing tissues underwent callus cell growth, but organogenesis was repressed. Transcriptome analysis revealed that several pathways depend on proper regulation of GhTCE1 expression, including lipid transfer pathway components, ROS homeostasis, and cell expansion. GhTCE1 bound to the promoters of the target genes GhLTP2 and GhLTP3, activating their expression synergistically, and the heterodimer TCE1-TCEE1 enhances this activity. GhLTP2- and GhLTP3-deficient tissues accumulated ROS and had arrested callus cell elongation, which was restored by ROS scavengers. These results reveal a unique regulatory network involving ROS and lipid transfer proteins, which act as potential ROS scavengers. This network acts as a switch between unorganized callus growth and organized development during in vitro dedifferentiation of cotton cells.

Histone ubiquitination controls organ size in cotton (Gossypium hirsutum).

Ubiquitination plays a vital role in modifying protein activity and destiny. Ub-conjugating enzyme E2 is one of the enzymes that participates in this precise process. There are at least 169 E2 proteins in the allotetraploid cotton (Gossypium hirsutum), but their function remains unknown. Here we identify an E2 gene GhUBC2L and show its positive role in cell proliferation and expansion. Complete knock-down of GhUBC2L in cotton resulted in retarded growth and reduced organ size. Conversely, overexpression of GhUBC2L promoted cotton growth, generating enlarged organs in size. Monoubiquitination of H2A and H2B was strongly impaired in GhUBC2L-suppressed cotton but slightly enhanced in GhUBC2L-overexpressed plant. GhUbox8, a U-box type E3 ligase protein, was found to interact with GhUBC2L both in vivo and in vitro, indicating their synergistical function in protein ubiquitination. Furthermore, GhUbox8 was shown to interact with a series of histone proteins, including histone H2A and H2B, indicating its potential monoubiquitination on H2A and H2B. Expression of genes relating to cell cycle and organ development were altered when the expression of GhUBC2L was changed. Our results show that GhUBC2L modulates histone monoubiquitination synergistically with GhUbox8 to regulate the expression of genes involved in organ development and cell cycle, thus controlling organ size in cotton. This research provides new insights into the role of protein ubiquitination in organ size control. Histone monoubiquitination plays an important role in plant development. Here, we identified an E2 enzyme GhUBC2L that modulates histone monoubiquitination synergistically with an E3 ligase GhUbox8 to mediate organ size control in cotton.

Genome-wide analysis of CBL and CIPK family genes in cotton: conserved structures with divergent interactions and expression.

Calcineurin B-like proteins (CBLs) interact with CBL-interacting protein kinases (CIPKs) to form complex molecular modules in response to diverse abiotic stresses. Although previous studies demonstrated that the CBL-CIPK networks play a crucial role in plants response to abiotic stresses, however, little is known about their functions in cotton. In the present study, a total of 22 GhCBL and 79 GhCIPK gene family members were identified in upland cotton (Gossypium hirsutum Linn). Synteny analysis revealed that most genes of GhCBL and GhCIPK exist in pairs between At sub-genome and Dt sub-genome. Interaction analysis between GhCBL and GhCIPK proteins by yeast two-hybrid (Y2H) suggested that the GhCBL-GhCIPK networks were complex, and exhibited functional redundancy in cotton. Quantitative expression analysis by public transcriptome datasets revealed that some GhCBL and GhCIPK genes are differentially expressed under abiotic stress treatments, and especially under drought stress. Our results not only contribute to understanding the structural features of GhCBL and GhCIPK genes but also provide the basis for in-depth functional studies of GhCBL-GhCIPK networks in stress response for plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (doi:10.1007/s12298-021-00943-1).

The Calcium Sensor CBL2 and Its Interacting Kinase CIPK6 Are Involved in Plant Sugar Homeostasis via Interacting with Tonoplast Sugar Transporter TST2.

Calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK)-mediated calcium signaling has been widely reported to function in plant development and various stress responses, particularly in ion homeostasis. Sugars are the most important primary metabolites, and thus sugar homeostasis requires precise regulation. Here, we describe a CBL2-CIPK6-Tonoplast-Localized Sugar Transporter2 (TST2) molecular module in cotton (Gossypium hirsutum) that regulates plant sugar homeostasis, in particular Glc homeostasis. GhCIPK6 is recruited to the tonoplast by GhCBL2 and interacts with the tonoplast-localized sugar transporter GhTST2. Overexpression of either GhCBL2, GhCIPK6, or GhTST2 was sufficient to promote sugar accumulation in transgenic cotton, whereas RNAi-mediated knockdown of GhCIPK6 expression or CRISPR-Cas9-mediated knockout of GhTST2 resulted in significantly decreased Glc content. Moreover, mutation of GhCBL2 or GhTST2 in GhCIPK6-overexpressing cotton reinstated sugar contents comparable to wild-type plants. Heterologous expression of GhCIPK6 in Arabidopsis (Arabidopsis thaliana) also promoted Glc accumulation, whereas mutation of AtTST1/2 in GhCIPK6-overexpressing Arabidopsis similarly reinstated wild-type sugar contents, thus indicating conservation of CBL2-CIPK6-TST2-mediated sugar homeostasis among different plant species. Our characterization of the molecular players behind plant sugar homeostasis may be exploited to improve sugar contents and abiotic stress resistance in plants.

High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system.

Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene-specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site-specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2-edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7-100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode-based high-throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1-edited T0 plants and it matched well with Sanger sequencing results. No off-target editing was detected at the potential off-target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.

Interaction between calcium and potassium modulates elongation rate in cotton fiber cells.

Calcium (Ca2+) is necessary for fiber cell development in cotton (Gossypium hirsutum), both as a cell wall structural component and for environmental signaling responses. It is also known that potassium (K+) plays a critical role in cotton fiber cell elongation. However, it is unclear whether Ca2+ integrates its activities with K+ to regulate fiber elongation. Here, we report the novel discovery that Ca2+ deficiency, when integrated with K+ signaling, promotes fiber elongation. Using inductively coupled plasma-mass spectrometry (ICP-MS), we determined dynamic profiles of the ionome in ovules and fibers at different developmental stages, and found that a high accumulation of macro-elements, but not Ca2+, was associated with longer fibers. Using an in vitro ovule culture system, we found that under Ca2+-deficient conditions, sufficient K+ (52 mM) rapidly induced ovule and fiber browning, while reduced K+ (2 or 27 mM) not only suppressed tissue browning but also altered fiber elongation. Reduced K+ also enhanced reactive oxygen species scavenging ability and maintained abscisic acid and jasmonic acid levels, which in turn compensated for Ca2+ deficiency. Ca2+ deficiency combined with reduced K+ (0 mM Ca2+ and 27 mM K+) produced longer fibers in cultured ovules, due to cell wall loosening by phytosulfokine (PSK), expansin (EXP), and xyloglucan endotransglycosylase/hydrolase (XTH), and an increase of the K+ content of fiber cells. Using transgenic cotton, we showed that the CBL-INTERACTING PROTEIN KINASE 6 (GhCIPK6) gene mediates the uptake of K+ under Ca2+-deficient conditions. This study establishes a new link between Ca2+, K+, and fiber elongation.

ROS Homeostasis Regulates Somatic Embryogenesis via the Regulation of Auxin Signaling in Cotton.

Somatic embryogenesis (S.E.) is a versatile model for understanding the mechanisms of plant embryogenesis and a useful tool for plant propagation. To decipher the intricate molecular program and potentially to control the parameters affecting the frequency of S.E., a proteomics approach based on two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF/TOF was used. A total of 149 unique differentially expressed proteins (DEPs) were identified at different stages of cotton S.E. compared with the initial control (0 h explants). The expression profile and functional annotation of these DEPs revealed that S.E. activated stress-related proteins, including several reactive oxygen species (ROS)-scavenging enzymes. Proteins implicated in metabolic, developmental, and reproductive processes were also identified. Further experiments were performed to confirm the role of ROS-scavenging enzymes, suggesting the involvement of ROS homeostasis during S.E. in cotton. Suppressing the expression of specifically identified GhAPX proteins resulted in the inhibition of dedifferentiation. Accelerated redifferentiation was observed in the suppression lines of GhAPXs or GhGSTL3 in parallel with the alteration of endogenous ascorbate metabolism and accumulation of endogenous H2O2 content. Moreover, disrupting endogenous redox homeostasis through the application of high concentrations of DPI, H2O2, BSO, or GSH inhibited the dedifferentiation of cotton explants. Mild oxidation induced through BSO treatment facilitated the transition from embryogenic calluses (ECs) to somatic embryos. Meanwhile, auxin homeostasis was altered through the perturbation of ROS homeostasis by chemical treatments or suppression of ROS-scavenging proteins, along with the activating/suppressing the transcription of genes related to auxin transportation and signaling. These results show that stress responses are activated during S.E. and may regulate the ROS homeostasis by interacting with auxin signaling.

Molecular cloning and functional characterization of a novel cotton CBL-interacting protein kinase gene (GhCIPK6) reveals its involvement in multiple abiotic stress tolerance in transgenic plants.

Plant CIPKs were specific Ser/Thr protein kinases, which were activated through interaction with calcineurin B-like protein (CBL) containing four EF hands for Ca(2+) binding. The CBL/CIPK complexes play an important role in signal transduction in biotic and abiotic stresses, as well as developmental processes. Here a Ser/Thr protein kinase gene (defined as GhCIPK6), which was isolated from RNA-Seq profile during cotton somatic embryogenesis in our previous research was characterized. The GhCIPK6 gene contains an ORF of 1296 bp that putatively encodes a polypeptide of 431 amino acids with a predicted molecular mass of 48.46 kDa and isoelectric point of 9.12. Sequence alignment analysis confirmed that GhCIPK6 has no intron, and it was homologous to AtCIPK6. Expression analysis of the GhCIPK6 suggested that they might function in diverse tissues, including styles and anthers but not fibers. In addition, expression of the GhCIPK6 gene was induced by salt, drought and ABA treatments. Overexpression of GhCIPK6 significantly enhances the tolerance to salt, drought and ABA stresses in transgenic Arabidopsis, indicating that GhCIPK6 acts as a positive regulator in response to salt and drought stress, and is supposed to be a potential candidate gene to improve stress tolerance by genetic manipulation in cotton and other crops.