GhMYB30-GhMUR3 affects fiber elongation and secondary wall thickening in cotton.

Xyloglucan, an important hemicellulose, plays a crucial role in maintaining cell wall structure and cell elongation. However, the effects of xyloglucan on cotton fiber development are not well understood. GhMUR3 encodes a xyloglucan galactosyltransferase that is essential for xyloglucan synthesis and is highly expressed during fiber elongation. In this study, we report that GhMUR3 participates in cotton fiber development under the regulation of GhMYB30. Overexpression GhMUR3 affects the fiber elongation and cell wall thickening. Transcriptome showed that the expression of genes involved in secondary cell wall synthesis was prematurely activated in OE-MUR3 lines. In addition, GhMYB30 was identified as a key regulator of GhMUR3 by Y1H, Dual-Luc, and electrophoretic mobility shift assay (EMSA) assays. GhMYB30 directly bound the GhMUR3 promoter and activated GhMUR3 expression. Furthermore, DAP-seq of GhMYB30 was performed to identify its target genes in the whole genome. The results showed that many target genes were associated with fiber development, including cell wall synthesis-related genes, BR-related genes, reactive oxygen species pathway genes, and VLCFA synthesis genes. It was demonstrated that GhMYB30 may regulate fiber development through multiple pathways. Additionally, GhMYB46 was confirmed to be a target gene of GhMYB30 by EMSA, and GhMYB46 was significantly increased in GhMYB30-silenced lines, indicating that GhMYB30 inhibited GhMYB46 expression. Overall, these results revealed that GhMUR3 under the regulation of GhMYB30 and plays an essential role in cotton fiber elongation and secondary wall thickening. Additionally, GhMYB30 plays an important role in the regulation of fiber development and regulates fiber secondary wall synthesis by inhibiting the expression of GhMYB46.

Comparative Physiological and Transcriptome Analysis Reveals Potential Pathways and Specific Genes Involved in Waterlogging Tolerance in Apple Rootstocks.

Apple (Malus × domestica Borkh.) is one of the most cultivated fruit crops in China. Apple trees frequently encounter waterlogging stress, mainly due to excess rainfall, soil compaction, or poor soil drainage, results in yellowing leaves and declined fruit quality and yield in some regions. However, the mechanism underlying the response to waterlogging has not been well elucidated. Therefore, we performed a physiological and transcriptomic analysis to examine the differential responses of two apple rootstocks (waterlogging-tolerant M. hupehensis and waterlogging-sensitive M. toringoides) to waterlogging stress. The results showed that M. toringoides displayed more severe leaf chlorosis during the waterlogging treatment than M. hupehensis. Compared with M. hupehensis, the more severe leaf chlorosis induced by waterlogging stress in M. toringoides was highly correlated with increased electrolyte leakage and superoxide radicals, hydrogen peroxide accumulation, and increased stomata closure. Interestingly, M. toringoides also conveyed a higher ethylene production under waterlogging stress. Furthermore, RNA-seq revealed that a total of 13,913 common differentially expressed genes (DEGs) were differentially regulated between M. hupehensis and M. toringoides under waterlogging stress, especially those DEGs involved in the biosynthesis of flavonoids and hormone signaling. This suggests a possible link of flavonoids and hormone signaling to waterlogging tolerance. Taken together, our data provide the targeted genes for further investigation of the functions, as well as for future molecular breeding of waterlogging-tolerant apple rootstocks.

The engineered CRISPR-Mb2Cas12a variant enables sensitive and fast nucleic acid-based pathogens diagnostics in the field.

Existing CRISPR/Cas12a-based diagnostic platforms offer accurate and vigorous monitoring of nucleic acid targets, but have the potential to be further optimized for more efficient detection. Here, we profiled 16 Cas12a orthologs, focusing on their trans-cleavage activity and their potential as diagnostic enzymes. We observed the Mb2Cas12a has more robust trans-cleavage activity than other orthologs, especially at lower temperatures. An engineered Mb2Cas12a-RRVRR variant presented robust trans-cleavage activity and looser PAM constraints. Moreover, we found the existing one-pot assay, which simultaneously performed Recombinase Polymerase Amplification (RPA) and Cas12a reaction in one system, resulted in the loss of single-base discrimination during diagnosis. Therefore, we designed a reaction vessel that physically separated the RPA and Cas12a steps while maintaining a closed system. This isolated but closed system made diagnostics more sensitive and specific and effectively prevented contamination. This shelved Mb2Cas12a-RRVRR variant-mediated assay detected various targets in less than 15 min and exhibited equal or greater sensitivity than qPCR when detecting bacterial pathogens, plant RNA viruses and genetically modified crops. Overall, our findings further improved the efficiency of the current CRISPR-based diagnostic system and undoubtedly have great potential for highly sensitive and specific detection of multiple sample types.

Long-distance mobile mRNA CAX3 modulates iron uptake and zinc compartmentalization.

Iron deficiency in plants can lead to excessive absorption of zinc; however, important details of this mechanism have yet to be elucidated. Here, we report that MdCAX3 mRNA is transported from the leaf to the root, and that MdCAX3 is then activated by MdCXIP1. Suppression of MdCAX3 expression leads to an increase in the root apoplastic pH, which is associated with the iron deficiency response. Notably, overexpression of MdCAX3 does not affect the apoplastic pH in a MdCXIP1 loss-of-function Malus baccata (Mb) mutant that has a deletion in the MdCXIP1 promoter. This deletion in Mb weakens MdCXIP1 expression. Co-expression of MdCAX3 and MdCXIP1 in Mb causes a decrease in the root apoplastic pH. Furthermore, suppressing MdCAX3 in Malus significantly reduces zinc vacuole compartmentalization. We also show that MdCAX3 activated by MdCXIP1 is not only involved in iron uptake, but also in regulating zinc detoxification by compartmentalizing zinc in vacuoles to avoid iron starvation-induced zinc toxicity. Thus, mobile MdCAX3 mRNA is involved in the regulation of iron and zinc homeostasis in response to iron starvation.

The MADS transcription factor GhFYF is involved in abiotic stress responses in upland cotton (Gossypium hirsutum L.).

Cotton is an important textile industry raw material crops, which plays a critical role in the development of society. MADS transcription factors (TFs) play a key role about the flowering time, flower development, and abiotic stress responses in plants, but little is known about their functions on abiotic stress in cotton. In this study, a MIKCC subfamily gene from cotton, GhFYF (FOREVER YOUNG FLOWER), was isolated and characterized. Our data showed that GhFYF localized to the nucleus. A ß-glucuronidase (GUS) activity assay revealed that the promoter of GhFYF was mainly expressed in the flower and seed of ProGhFYF::GUS transgenic A. thaliana plants. The GUS staining of flowers and seeds was deepened after drought, salt treatment, and the expression level of the GUS gene and corresponding stress genes AtERD10, AtAnnexin1 are up-regulated in the inflorescence. Overexpression GhFYF in A. thaliana could promote the seed germination and growth under different salt concentrations, and determin the proline content. Yeast two-hybrid (Y2H) assays showed that GhFYF interacted with the HAD-like protein GhGPP2, which has responds to abiotic stress. Our findings indicate that GhFYF is involved in abiotic stress responses, especially for salt stress. This work establishes a solid foundation for further functional analysis of the GhFYF gene in cotton.

Silencing of GhKEA4 and GhKEA12 Revealed Their Potential Functions Under Salt and Potassium Stresses in Upland Cotton.

The K+ efflux antiporter (KEA) mediates intracellular K+ and H+ homeostasis to improve salt tolerance in plants. However, the knowledge of KEA gene family in cotton is largely absent. In the present study, 8, 8, 15, and 16 putative KEA genes were identified in Gossypium arboreum, G. raimondii, G. hirsutum, and G. barbadense, respectively. These KEA genes were classified into three subfamilies, and members from the same subfamilies showed similar motif compositions and gene structure characteristics. Some hormone response elements and stress response elements were identified in the upstream 2000 bp sequence of GhKEAs. Transcriptome data showed that most of the GhKEAs were highly expressed in roots and stems. The quantificational real-time polymerase chain reaction (qRT-PCR) results showed that most of the GhKEAs responded to low potassium, salt and drought stresses. Virus-induced gene silencing (VIGS) experiments demonstrated that under salt stress, after silencing genes GhKEA4 and GhKEA12, the chlorophyll content, proline content, soluble sugar content, peroxidase (POD) activity and catalase (CAT) activity were significantly decreased, and the Na+/K+ ratio was extremely significantly increased in leaves, leading to greater salt sensitivity. Under high potassium stress, cotton plants silenced for the GhKEA4 could still maintain a more stable Na+ and K+ balance, and the activity of transporting potassium ions from roots into leaves was reduced silenced for GhKEA12. Under low potassium stress, silencing the GhKEA4 increased the activity of transporting potassium ions to shoots, and silencing the GhKEA12 increased the ability of absorbing potassium ions, but accumulated more Na+ in leaves. These results provided a basis for further studies on the biological roles of KEA genes in cotton development and adaptation to stress conditions.

RBP differentiation contributes to selective transmissibility of OPT3 mRNAs.

Long-distance mobile mRNAs play key roles in gene regulatory networks that control plant development and stress tolerance. However, the mechanisms underlying species-specific delivery of mRNA still need to be elucidated. Here, the use of grafts involving highly heterozygous apple (Malus) genotypes allowed us to demonstrate that apple (Malus domestica) oligopeptide transporter3 (MdOPT3) mRNA can be transported over a long distance, from the leaf to the root, to regulate iron uptake; however, the mRNA of Arabidopsis (Arabidopsis thaliana) oligopeptide transporter 3 (AtOPT3), the MdOPT3 homolog from A. thaliana, does not move from shoot to root. Reciprocal heterologous expression of the two types of mRNAs showed that the immobile AtOPT3 became mobile and moved from the shoot to the root in two woody species, Malus and Populus, while the mobile MdOPT3 became immobile in two herbaceous species, A. thaliana and tomato (Solanum lycopersicum). Furthermore, we demonstrated that the different transmissibility of OPT3 in A. thaliana and Malus might be caused by divergence in RNA-binding proteins between herbaceous and woody plants. This study provides insights into mechanisms underlying differences in mRNA mobility and validates the important physiological functions associated with this process.

Genome-Wide Identification and Functional Investigation of 1-Aminocyclopropane-1-carboxylic Acid Oxidase (ACO) Genes in Cotton.

ACO is one of the rate-limiting enzymes in the biosynthesis of ethylene, and it plays a critical role in the regulation of plant growth and development. However, the function of ACO genes in cotton is not well studied. In this study, a total of 332 GhACOs, 187 GaACOs, and 181 GrACOs were identified in G. hirsutum, G. arboretum, and G. raimondii, respectively. Gene duplication analysis showed that whole-genome duplication (WGD) and tandem duplication were the major forces driving the generation of cotton ACO genes. In the promoters of GhACOs, there were cis-acting elements responding to stress, phytohormones, light, and circadian factors, indicating the possible involvement of GhACOs in these processes. Expression and co-expression analyses illustrated that most GhACOs were not only widely expressed in various tissues but also coexpressed with other genes in response to salt and drought stress. GhACO106_At overexpression in Arabidopsis promoted flowering and increased salt tolerance. These results provide a comprehensive overview of the ACO genes of cotton and lay the foundation for subsequent functional studies of these genes.

GhLUX1 and GhELF3 Are Two Components of the Circadian Clock That Regulate Flowering Time of Gossypium hirsutum.

Photoperiod is an important external factor that regulates flowering time, the core mechanism of which lies in the circadian clock-controlled expression of FLOWERING LOCUS T (FT) and its upstream regulators. However, the roles of the circadian clock in regulating cotton flowering time are largely unknown. In this study, we cloned two circadian clock genes in cotton, GhLUX1 and GhELF3. The physicochemical and structural properties of their putative proteins could satisfy the prerequisites for the interaction between them, which was proved by yeast two-hybrid (Y2H) and Bimolecular Fluorescent Complimentary (BiFC) assays. Phylogenetic analysis of LUXs and ELF3s indicated that the origin of LUXs was earlier than that of ELF3s, but ELF3s were more divergent and might perform more diverse functions. GhLUX1, GhELF3, GhCOL1, and GhFT exhibited rhythmic expression and were differentially expressed in the early flowering and late-flowering cotton varieties under different photoperiod conditions. Both overexpression of GhLUX1 and overexpression of GhELF3 in Arabidopsis delayed flowering probably by changing the oscillation phases and amplitudes of the key genes in the photoperiodic flowering pathway. Both silencing of GhLUX1 and silencing of GhELF3 in cotton increased the expression of GhCOL1 and GhFT and resulted in early flowering. In summary, the circadian clock genes were involved in regulating cotton flowering time and could be the candidate targets for breeding early maturing cotton varieties.

GhGPAT12/25 Are Essential for the Formation of Anther Cuticle and Pollen Exine in Cotton (Gossypium hirsutum L.).

Glycerol-3-phosphate acyltransferases (GPATs), critical for multiple biological processes like male fertility, have been extensively characterized. However, their precise functions and underlying regulatory mechanism in cotton anther development are unclear. This research demonstrated the importance of GhGPAT12/25 (a paralogs pair on A12/D12 sub-chromosome of cotton) to regulate the degradation of tapetum, anther cuticle formation, and pollen exine development. GhGPAT12 and GhGPAT25 exhibited specifically detected transcripts in tapetum and pollen exine during the early anther developmental stages. GhGPAT12/25 are sn-2 glycerol-3-phosphate acyltransferases and can transfer the acyl group of palmitoyl-CoA to glycerol-3-phosphate (G3P). CRISPR/Cas9-mediated knockout identified the functional redundancy of GhGPAT12 and GhGPAT25. Knockout of both genes caused completely male sterility associated with abnormal anther cuticle, swollen tapetum, and inviable microspores with defective exine and irregular unrestricted shape. RNA-seq analysis showed that the loss of function of GhGPAT12/25 affects the processes of wax metabolic, glycerol monomer biosynthesis, and transport. Consistently, cuticular waxes were dramatically reduced in mutant anthers. Yeast one-hybrid system (Y1H), virus-induced gene silencing (VIGS), and dual-luciferase (LUC) assays illustrated that GhMYB80s are likely to directly activate the expression of GhGPAT12/25. This study provides important insights for revealing the regulatory mechanism underlying anther development in cotton.

High-resolution temporal dynamic transcriptome landscape reveals a GhCAL-mediated flowering regulatory pathway in cotton (Gossypium hirsutum L.).

The transition from vegetative to reproductive growth is very important for early maturity in cotton. However, the genetic control of this highly dynamic and complex developmental process remains unclear. A high-resolution tissue- and stage-specific transcriptome profile was generated from six developmental stages using 72 samples of two early-maturing and two late-maturing cotton varieties. The results of histological analysis of paraffin sections showed that flower bud differentiation occurred at the third true leaf stage (3TLS) in early-maturing varieties, but at the fifth true leaf stage (5TLS) in late-maturing varieties. Using pairwise comparison and weighted gene co-expression network analysis, 5312 differentially expressed genes were obtained, which were divided into 10 gene co-expression modules. In the MElightcyan module, 46 candidate genes regulating cotton flower bud differentiation were identified and expressed at the flower bud differentiation stage. A novel key regulatory gene related to flower bud differentiation, GhCAL, was identified in the MElightcyan module. Anti-GhCAL transgenic cotton plants exhibited late flower bud differentiation and flowering time. GhCAL formed heterodimers with GhAP1-A04/GhAGL6-D09 and regulated the expression of GhAP1-A04 and GhAGL6-D09. GhAP1-A04- and GhAGL6-D09-silenced plants also showed significant late flowering. Finally, we propose a new flowering regulatory pathway mediated by GhCAL. This study elucidated the molecular mechanism of cotton flowering regulation and provides good genetic resources for cotton early-maturing breeding.

Evolution and Stress Responses of CLO Genes and Potential Function of the GhCLO06 Gene in Salt Resistance of Cotton.

The caleosin (CLO) protein family displays calcium-binding properties and plays an important role in the abiotic stress response. Here, a total of 107 CLO genes were identified in 15 plant species, while no CLO genes were detected in two green algal species. Evolutionary analysis revealed that the CLO gene family may have evolved mainly in terrestrial plants and that biological functional differentiation between species and functional expansion within species have occurred. Of these, 56 CLO genes were identified in four cotton species. Collinearity analysis showed that CLO gene family expansion mainly occurred through segmental duplication and whole-genome duplication in cotton. Sequence alignment and phylogenetic analysis showed that the CLO proteins of the four cotton species were mainly divided into two types: H-caleosins (class I) and L-caleosins (class II). Cis-acting element analysis and quantitative RT-PCR (qRT-PCR) suggested that GhCLOs might be regulated by abscisic acid (ABA) and methyl jasmonate (MeJA). Moreover, transcriptome data and qRT-PCR results revealed that GhCLO genes responded to salt and drought stresses. Under salt stress, gene-silenced plants (TRV: GhCLO06) showed obvious yellowing and wilting, higher malondialdehyde (MDA) content accumulation, and significantly lower activities of superoxide dismutase (SOD) and peroxidase (POD), indicating that GhCLO06 plays a positive regulatory role in cotton salt tolerance. In gene-silenced plants (TRV: GhCLO06), ABA-related genes (GhABF2, GhABI5, and GhNAC4) were significantly upregulated after salt stress, suggesting that the regulation of salt tolerance may be related to the ABA signaling pathway. This research provides an important reference for further understanding and analyzing the molecular regulatory mechanism of CLOs for salt tolerance.

The MADS transcription factor GhAP1.7 coordinates the flowering regulatory pathway in upland cotton (Gossypium hirsutum L.).

MADS-box gene family plays an important role in the molecular regulatory network of flower development. APETALA1 (AP1), a MADS-box gene, plays an important role in the development of flower organs. Although many studies about MADS-box family genes have been reported, the function of AP1 is still not clear in cotton. In this study, GhAP1.7 (Gh_D03G0922), a candidate gene for cotton flower time and plant height obtained from our previous studies, was cloned from CCRI50 cotton variety and functionally characterized. Subcellular localization demonstrated that GhAP1.7 was located in nucleus. Infection test of Arabidopsis revealed that GhAP1.7 could cause precocious flowering and virus-induced gene silence (VIGS) assay demonstrated that GhAP1.7 could lead to delayed flowering of cotton plants. Yeast one-hybrid assays and transient dual-luciferase assays suggested that floral meristem identity control gene LEAFY (LFY) can bind the promoter of GhAP1.7 and negatively regulate it. Our research indicated that GhAP1.7 might work as a positive regulator in plant flowering. Moreover, GhAP1.7 may negatively regulated by GhLFY in the regulatory pathways. This work laid the foundation for subsequent functional studies of GhAP1.7.

A comprehensive analysis of cotton VQ gene superfamily reveals their potential and extensive roles in regulating cotton abiotic stress.

BACKGROUND: Valine-glutamine (VQ) motif-containing proteins play important roles in plant growth, development and abiotic stress response. For many plant species, the VQ genes have been identified and their functions have been described. However, little is known about the origin, evolution, and functions (and underlying mechanisms) of the VQ family genes in cotton. RESULTS: In this study, we comprehensively analyzed the characteristics of 268 VQ genes from four Gossypium genomes and found that the VQ proteins evolved into 10 clades, and each clade had a similar structural and conservative motif. The expansion of the VQ gene was mainly through segmental duplication, followed by dispersal. Expression analysis revealed that many GhVQs might play important roles in response to salt and drought stress, and GhVQ18 and GhVQ84 were highly expressed under PEG and salt stress. Further analysis showed that GhVQs were co-expressed with GhWRKY transcription factors (TFs), and microRNAs (miRNAs) could hybridize to their cis-regulatory elements. CONCLUSIONS: The results in this study broaden our understanding of the VQ gene family in plants, and the analysis of the structure, conserved elements, and expression patterns of the VQs provide a solid foundation for exploring their specific functions in cotton responding to abiotic stresses. Our study provides significant insight into the potential functions of VQ genes in cotton.

Genome-wide identification and expression patterns analysis of the RPD3/HDA1 gene family in cotton.

BACKGROUND: Histone deacetylases (HDACs) catalyze histone deacetylation and suppress gene transcription during various cellular processes. Within the superfamily of HDACs, RPD3/HDA1-type HDACs are the most studied, and it is reported that RPD3 genes play crucial roles in plant growth and physiological processes. However, there is a lack of systematic research on the RPD3/HDA1 gene family in cotton. RESULTS: In this study, genome-wide analysis identified 9, 9, 18, and 18 RPD3 genes in Gossypium raimondii, G. arboreum, G. hirsutum, and G. barbadense, respectively. This gene family was divided into 4 subfamilies through phylogenetic analysis. The exon-intron structure and conserved motif analysis revealed high conservation in each branch of the cotton RPD3 genes. Collinearity analysis indicated that segmental duplication was the primary driving force during the expansion of the RPD3 gene family in cotton. There was at least one presumed cis-element related to plant hormones in the promoter regions of all GhRPD3 genes, especially MeJA- and ABA-responsive elements, which have more members than other hormone-relevant elements. The expression patterns showed that most GhRPD3 genes had relatively high expression levels in floral organs and performed higher expression in early-maturity cotton compared with late-maturity cotton during flower bud differentiation. In addition, the expression of GhRPD3 genes could be significantly induced by one or more abiotic stresses as well as exogenous application of MeJA or ABA. CONCLUSIONS: Our findings reveal that GhRPD3 genes may be involved in flower bud differentiation and resistance to abiotic stresses, which provides a basis for further functional verification of GhRPD3 genes in cotton development and a foundation for breeding better early-maturity cotton cultivars in the future.

Genome-wide identification and characterization of multiple C2 domains and transmembrane region proteins in Gossypium hirsutum.

BACKGROUND: Multiple C2 domains and transmembrane region proteins (MCTPs) may act as transport mediators of other regulators. Although increased number of MCTPs in higher plants implies their diverse and specific functions in plant growth and development, only a few plant MCTPs have been studied and no study on the MCTPs in cotton has been reported. RESULTS: In this study, we identified 31 MCTPs in G. hirsutum, which were classified into five subfamilies according to the phylogenetic analysis. GhMCTPs from subfamily V exhibited isoelectric points (pIs) less than 7, whereas GhMCTPs from subfamily I, II, III and IV exhibited pIs more than 7.5, implying their distinct biological functions. In addition, GhMCTPs within subfamily III, IV and V exhibited more diverse physicochemical properties, domain architectures and expression patterns than GhMCTPs within subfamily I and II, suggesting that GhMCTPs within subfamily III, IV and V diverged to perform more diverse and specific functions. Analyses of conserved motifs and pIs indicated that the N-terminus was more divergent than the C-terminus and GhMCTPs' functional divergence might be mainly contributed by the N-terminus. Furthermore, yeast two-hybrid assay indicated that the N-terminus was responsible to interact with target proteins. Phylogenetic analysis classified multiple N-terminal C2 domains into four subclades, suggesting that these C2 domains performed different molecular functions in mediating the transport of target proteins. CONCLUSIONS: Our systematic characterization of MCTPs in G. hirsutum will provide helpful information to further research GhMCTPs' molecular roles in mediating other regulators' transport to coordinate growth and development of various cotton tissues.

Pectate lyase-like Gene GhPEL76 regulates organ elongation in Arabidopsis and fiber elongation in cotton.

Pectate lyases (PELs) play important roles in plant growth and development, mainly by degrading the pectin in primary cell walls. However, the role of PELs in cotton fiber elongation, which also involves changes in cellular structure and components, is poorly understood. Therefore, we aimed to isolate and characterize GhPEL76, as we suspected it to contribute to the regulation of fiber elongation. Expression analysis (qRT-PCR) revealed that GhPEL76 is predominately expressed in cotton fiber, with significantly different expression levels in long- and short-fiber cultivars, and that GhPEL76 expression is responsive to gibberellic acid and indoleacetic acid treatment. Furthermore, GhPEL76 promoter-driven ß-glucuronidase activity was detected in the roots, hypocotyls, and leaves of transgenic Arabidopsis plants, and the overexpression of GhPEL76 in transgenic Arabidopsis promoted the elongation of several organs, including petioles, hypocotyls, primary roots, and trichomes. Additionally, the virus-induced silencing of GhPEL76 in cotton reduced fiber length, and both yeast one-hybrid and transient dual-luciferase assays suggested that GhbHLH13, a bHLH transcription factor that is up-regulated during fiber elongation, activates GhPEL76 expression by binding to the G-box of the GhPEL76 promoter region. Therefore, these results suggest GhPEL76 positively regulates fiber elongation and provide a basis for future studies of cotton fiber development.

A long non-coding apple RNA, MSTRG.85814.11, acts as a transcriptional enhancer of SAUR32 and contributes to the Fe-deficiency response.

Iron (Fe) is an essential plant nutrient and its deficiency typically limits plant growth. Long non-coding (lnc) RNAs are involved in adaptive responses to nutrient stress; however, it is not known whether they function in the regulation of the canonical Fe-deficiency response. The expression of Malus domestica (apple) lncRNA MSTRG.85814 is induced by Fe deficiency, as identified by high-throughput strand-specific RNA-seq analysis of an apple homograft system. MSTRG.85814 has a complex structure, with 13 predicted RNA sequence variants, four of which are upregulated in the roots of plants experiencing Fe deficiency. We found that one MSTRG.85814 splice variant (MSTRG.85814.11) positively modulated its cis target mRNA derived from the small auxin upregulated gene SAUR32. This in turn promoted the expression of SAUR32 and caused an increase in the expression of a plasma membrane proton ATPase, AHA10. Using a pH imaging technique, a significant decrease in the apoplastic pH was observed to occur in the root tips of MSTRG.85814.11 or SAUR32-overexpressing apple plants. Thus MSTRG.85814.11 was shown to positively promote SAUR32 expression, which then activated proton extrusion involved in the Fe-deficiency response. These results reveal a mechanism by which lncRNA promotes environmental Fe-deficiency stress adaption.

Genome-wide identification and characterization of TALE superfamily genes in cotton reveals their functions in regulating secondary cell wall biosynthesis.

BACKGROUND: Cotton fiber length and strength are both key traits of fiber quality, and fiber strength (FS) is tightly correlated with secondary cell wall (SCW) biosynthesis. The three-amino-acid-loop-extension (TALE) superclass homeoproteins are involved in regulating diverse biological processes in plants, and some TALE members has been identified to play a key role in regulating SCW formation. However, little is known about the functions of TALE members in cotton (Gossypium spp.). RESULTS: In the present study, based on gene homology, 46, 47, 88 and 94 TALE superfamily genes were identified in G. arboreum, G. raimondii, G. barbadense and G. hirsutum, respectively. Phylogenetic and evolutionary analysis showed the evolutionary conservation of two cotton TALE families (including BEL1-like and KNOX families). Gene structure analysis also indicated the conservation of GhTALE members under selection. The analysis of promoter cis-elements and expression patterns suggested potential transcriptional regulation functions in fiber SCW biosynthesis and responses to some phytohormones for GhTALE proteins. Genome-wide analysis of colocalization of TALE transcription factors with SCW-related QTLs revealed that some BEL1-like genes and KNAT7 homologs may participate in the regulation of cotton fiber strength formation. Overexpression of GhKNAT7-A03 and GhBLH6-A13 significantly inhibited the synthesis of lignocellulose in interfascicular fibers of Arabidopsis. Yeast two-hybrid (Y2H) experiments showed extensive heteromeric interactions between GhKNAT7 homologs and some GhBEL1-like proteins. Yeast one-hybrid (Y1H) experiments identified the upstream GhMYB46 binding sites in the promoter region of GhTALE members and defined the downstream genes that can be directly bound and regulated by GhTALE heterodimers. CONCLUSION: We comprehensively identified TALE superfamily genes in cotton. Some GhTALE members are predominantly expressed during the cotton fiber SCW thicking stage, and may genetically correlated with the formation of FS. Class II KNOX member GhKNAT7 can interact with some GhBEL1-like members to form the heterodimers to regulate the downstream targets, and this regulatory relationship is partially conserved with Arabidopsis. In summary, this study provides important clues for further elucidating the functions of TALE genes in regulating cotton growth and development, especially in the fiber SCW biosynthesis network, and it also contributes genetic resources to the improvement of cotton fiber quality.

Genome-Wide Identification and Characterization of Glycosyltransferase Family 47 in Cotton.

The glycosyltransferase (GT) 47 family is involved in the biosynthesis of xylose, pectin and xyloglucan and plays a significant role in maintaining the normal morphology of the plant cell wall. However, the functions of GT47s are less well known in cotton. In the present study, a total of 53, 53, 105 and 109 GT47 genes were detected by genome-wide identification in Gossypium arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively. All the GT47s were classified into six major groups via phylogenetic analysis. The exon/intron structure and protein motifs indicated that each branch of the GT47 genes was highly conserved. Collinearity analysis showed that GT47 gene family expansion occurred in Gossypium spp. mainly through whole-genome duplication and that segmental duplication mainly promoted GT47 gene expansion within the A and D subgenomes. The Ka/Ks values suggested that the GT47 gene family has undergone purifying selection during the long-term evolutionary process. Transcriptomic data and qRT-PCR showed that GhGT47 genes exhibited different expression patterns in each tissue and during fiber development. Our results suggest that some genes in the GhGT47 family might be associated with fiber development and the abiotic stress response, which could promote further research involving functional analysis of GT47 genes in cotton.