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Cotton is one of the most important economic and fiber crops in the world. KNOX is one class of universal transcription factors, which plays important roles in plant growth and development as well as response to different stresses. Although there are many researches on KNOXs in other plant species, there are few reports on cotton. In this study, we systematically and comprehensively identified all KNOX genes in upland cotton and its two ancestral species; we also studied their functions by employing RNA-seq analysis and virus-induced gene silence (VIGS). A total of 89 KNOX genes were identified from three cotton species. Among them, 44 were from upland cotton, 22 and 23 were found in its ancestral species G. raimondii and G. arboreum, respectively. Plant polyploidization and domestication play a selective force driving KNOX gene evolution. Phylogenetic analysis displayed that KNOX genes were evolved into three Classes. The intron length and exon number differed in each Class. Transcriptome data showed that KNOX genes of Class II were widely expressed in multiple tissues, including fiber. The majority of KNOX genes were induced by different abiotic stresses. Additionally, we found multiple cis-elements related to stress in the promoter region of KNOX genes. VIGS silence of GhKNOX4-A and GhKNOX22-D genes showed significant growth and development effect in cotton seedlings under salt and drought treatments. Both GhKNOX4-A and GhKNOX22-D regulated plant tolerance; silencing both genes induced oxidative stresses, evidenced by reduced SOD activity and induced leave cell death, and also enhanced stomatal open and water loss. Thus, GhKNOX4-A and GhKNOX22-D may contribute to drought response by regulating stomata opening and oxidative stresses., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.)

Plant specific transcription factors, SQUAMOSA promoter-binding protein-like (SPL), are involved in many biological processes. However, no systematical study has been reported in cotton. In this study, a total of 177 SPL genes were identified, including 29, 30, 59 and 59 SPLs in Gossypium arboreum, G. raimondii, G. barbadense, and G. hirsutum, respectively. These SPL genes were classified into eight phylogenetical groups. The gene structure, conserved motif, and clustering were highly conserved within each orthologs. Two zinc finger-like structures (Cys3His and Cys2HisCys) and NLS segments were existed in all GrSPLs. Segmental duplications play important roles in SPL family expansion, with 20 genes involved in segmental duplications and 2 in tandem duplications, and ten ortholog pairs in syntenic regions between G. raimondii and A. thaliana. Several putative cis-elements, involved in light, stresses and phytohormones response, were found in the promoter regions of GhSPLs, suggesting that plant responses to those environmental changes may be induced through targeting SPL transcription factors. RNA-seq analysis shows that SPL genes were differentially expressed in cotton; some were highly expressed during fiber initiation and early development. Comparing with other plants, SPL genes show subfunctionalization, lost and/or gain functions in cotton during long-term domestication and evolution.

Triclosan (TCS), an antimicrobial chemical with potential endocrine-disrupting properties, may pose a risk to early embryonic development and cellular homeostasis during adulthood. Here, we show that TCS induces toxicity in both the nematode C. elegans and human mesenchymal stem cells (hMSCs) by disrupting the SKN-1/Nrf2-mediated oxidative stress response. Specifically, TCS exposure affected C. elegans survival and hMSC proliferation in a dose-dependent manner. Cellular analysis showed that TCS inhibited the nuclear localization of SKN-1/Nrf2 and the expression of its target genes, which were associated with oxidative stress response. Notably, TCS-induced toxicity was significantly reduced by either antioxidant treatment or constitutive SKN-1/Nrf2 activation. As Nrf2 is strongly associated with aging and chemoresistance, these findings will provide a novel approach to the identification of therapeutic targets and disease treatment.

TCP proteins are plant-specific transcription factors implicated to perform a variety of physiological functions during plant growth and development. In the current study, we performed for the first time the comprehensive analysis of TCP gene family in a diploid cotton species, Gossypium arboreum, including phylogenetic analysis, chromosome location, gene duplication status, gene structure and conserved motif analysis, as well as expression profiles in fiber at different developmental stages. Our results showed that G. arboreum contains 36 TCP genes, distributing across all of the thirteen chromosomes. GaTCPs within the same subclade of the phylogenetic tree shared similar exon/intron organization and motif composition. In addition, both segmental duplication and whole-genome duplication contributed significantly to the expansion of GaTCPs. Many these TCP transcription factor genes are specifically expressed in cotton fiber during different developmental stages, including cotton fiber initiation and early development. This suggests that TCP genes may play important roles in cotton fiber development.

Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play versatile functions in multiple aspects of plant growth and development. However, no systematical study has been performed in cotton. In this study, we performed for the first time the genome-wide identification and expression analysis of the TCP transcription factor family in Gossypium raimondii. A total of 38 non-redundant cotton TCP encoding genes were identified. The TCP transcription factors were divided into eleven subgroups based on phylogenetic analysis. Most TCP genes within the same subfamily demonstrated similar exon and intron organization and the motif structures were highly conserved among the subfamilies. Additionally, the chromosomal distribution pattern revealed that TCP genes were unevenly distributed across 11 out of the 13 chromosomes; segmental duplication is a predominant duplication event for TCP genes and the major contributor to the expansion of TCP gene family in G. raimondii. Moreover, the expression profiles of TCP genes shed light on their functional divergence.

SUMMARY: The characterization of cis‐regulatory DNA elements (CREs) is essential for deciphering the regulation of gene expression in eukaryotes. Although there have been endeavors to identify CREs in plants, the properties of CREs in polyploid genomes are still largely unknown. Here, we conducted the genome‐wide identification of DNase I‐hypersensitive sites (DHSs) in leaf and stem tissues of the auto‐octoploid species Saccharum officinarum. We revealed that DHSs showed highly similar distributions in the genomes of these two S. officinarum tissues. Notably, we observed that approximately 74% of DHSs were located in distal intergenic regions, suggesting considerable differences in the abundance of distal CREs between S. officinarum and other plants. Leaf‐ and stem‐dependent transcriptional regulatory networks were also developed by mining the binding motifs of transcription factors (TFs) from tissue‐specific DHSs. Four TEOSINTE BRANCHED 1, CYCLOIDEA, and PCF1 (TCP) TFs (TCP2, TCP4, TCP7, and TCP14) and two ethylene‐responsive factors (ERFs) (ERF109 and ERF03) showed strong causal connections with short binding distances from each other, pointing to their possible roles in the regulatory networks of leaf and stem development. Through functional validation in transiently transgenic protoplasts, we isolate a set of tissue‐specific promoters. Overall, the DHS maps presented here offer a global view of the potential transcriptional regulatory elements in polyploid sugarcane and can be expected to serve as a valuable resource for both transcriptional network elucidation and genome editing in sugarcane breeding. Significance Statement: Whole genome‐wide open chromatin maps were constructed in the auto‐octoploid species Saccharum officinarum. We revealed that distal open chromatin was prevalent in sugarcane. Leaf‐ and stem‐specific promoters were isolated, which can be expected to serve as a valuable resource for both transcriptional network elucidation and genome editing in sugarcane breeding. [ABSTRACT FROM AUTHOR]

We identified 102, 51 and 51 proteins encoded by the trihelix genes in Gossypium hirsutum, Gossypium arboreum and Gossypiumraimondii, respectively. RNA sequence data and real‐time quantitative polymerase chain reaction analysis showed that Gh_A05G2067 (GT‐2) was highly upregulated under drought and salt stress conditions. Transient expression of GT‐2‐green fluorescent protein fusion protein in protoplast showed that GT‐2 was localized in the nucleus. The overexpression of GT‐2 conferred an enhanced drought tolerance to cotton, with lower malondialdehyde, hydrogen peroxide contents and higher reactive oxygen scavenging enzyme activities. Moreover, chlorophyll content, relative leaf water content (RLWC), excised leaf water loss (ELWL) and cell membrane stability (CMS) were relatively stable in the GT‐2‐overexpressed lines compared to wild‐type (WT). Similarly, stress‐responsive genes RD29A, SOS1, ABF4 and CBL1 were highly upregulated in the GT‐2‐overexpressed lines but were significantly downregulated in WT. In addition, the GT‐2‐silenced cotton plants exhibited a high level of oxidation injury, due to high levels of oxidant enzymes, in addition to negative effects on CMS, ELWL, RLWC and chlorophyll content. These results mark the foundation for future exploration of the trihelix genes in cotton, with an aim of developing more resilient, versatile and highly tolerant cotton genotypes. [ABSTRACT FROM AUTHOR]

Auxin response factors (ARFs) are recently discovered transcription factors that bind with auxin response elements (AuxRE, TGTCTC) to regulate the expression of early auxin-responsive genes. To our knowledge, the ARF gene family has never been characterized in cotton, the most important fiber crop in the world. In this study, a total of 35 ARF genes, named as GrARFs, were identified in a diploid cotton species Gossypium raimondii. The 35 ARF genes were located in 12 of the 13 cotton chromosomes; the intron/exon distribution of the GrARF genes was similar among sister pairs, whereas the divergence of some GrARF genes suggests the possibility of functional diversification. Our results show that the middle domains of nine GrARF proteins rich in glutamine (Q) are activators, while 26 other GrARF proteins rich in proline (P), serine (S), and threonine (T) are repressors. Our results also show that the expression of GrARF genes is diverse in different tissues. The expression of GrARF1 was significantly higher in leaves, whereas GrARF2a had higher expression level in shoots, which implicates different roles in the tested tissues. The GrARF11 has a higher expression level in buds than that in leaves, while GrARF19.2 shows contrasting expression patterns, having higher expression in leaves than that in buds. This suggests that they play different roles in leaves and buds. During long-term evolution of G. raimondii, some ARF genes were lost and some arose. The identification and characterization of the ARF genes in G. raimondii elucidate its important role in cotton that ARF genes regulate the development of flower buds, sepals, shoots, and leaves. [ABSTRACT FROM AUTHOR]

Micro RNAs (mi RNAs) have been found to be differentially expressed during cotton fibre development. However, which specific mi RNAs and how they are involved in fibre development is unclear. Here, using deep sequencing, 65 conserved mi RNA families were identified and 32 families were differentially expressed between leaf and ovule. At least 40 mi RNAs were either leaf or ovule specific, whereas 62 mi RNAs were shared in both leaf and ovule. q RT- PCR confirmed these mi RNAs were differentially expressed during fibre early development. A total of 820 genes were potentially targeted by the identified mi RNAs, whose functions are involved in a series of biological processes including fibre development, metabolism and signal transduction. Many predicted mi RNA-target pairs were subsequently validated by degradome sequencing analysis. GO and KEGG analyses showed that the identified mi RNAs and their targets were classified to 1027 GO terms including 568 biological processes, 324 molecular functions and 135 cellular components and were enriched to 78 KEGG pathways. At least seven unique mi RNAs participate in trichome regulatory interaction network. Eleven trans-acting si RNA (tasi RNA) candidate genes were also identified in cotton. One has never been found in other plant species and two of them were derived from MYB and ARF, both of which play important roles in cotton fibre development. Sixteen genes were predicted to be tasi RNA targets, including sucrose synthase and MYB2. Together, this study discovered new mi RNAs in cotton and offered evidences that mi RNAs play important roles in cotton ovule/fibre development. The identification of tasi RNA genes and their targets broadens our understanding of the complicated regulatory mechanism of mi RNAs in cotton. [ABSTRACT FROM AUTHOR]

Micro RNAs (mi RNAs) are an important class of small regulatory RNAs. The goal of this study was to analyse stress-responsive mi RNAs in switchgrass ( Panicum virgatum), the emerging biofuel crop, to facilitate choosing gene targets for improving biomass and biofuel yield. After sequencing three small RNA libraries constructed from control, salt- and drought-treated switchgrass using Illumina sequencing technology, we identified 670 known mi RNA families from a total of more than 50 million short reads. A total of 273 mi RNAs were identified with precursors: 126 conserved mi RNAs and 147 novel mi RNAs. Of them, 265 mi RNAs were found to have their opposite sequences (mi RNA*) with 2-nt overhang on the 3′ end. Of them, 194 were detected in switchgrass transcriptome sequences generated from 31 high-throughput RNA sequencing ( RNA-Seq) data sets in NCBI. Many mi RNAs were differentially or uniquely expressed during salinity or drought stress treatment. We also discovered 11 mi RNA clusters containing 29 mi RNAs. These identified mi RNAs potentially targeted 28 549 genes with a various function, including transcription factors, stress-response proteins and cellulose biosynthesis-related proteins. Gene ontology ( GO) and Kyoto Encyclopedia of Genes and Genomes ( KEGG) analysis showed that the identified mi RNAs and their targets were classified to 3779 GO terms including 1534 molecular functions, 1851 biological processes and 394 cellular components and were enriched to 147 KEGG pathways. Interestingly, 195 mi RNA families and 450 targets were involved in the biosynthesis pathways of carbon, glucose, starch, fatty acid and lignin and in xylem formation, which could aid in designing next-generation switchgrass for biomass and biofuel. [ABSTRACT FROM AUTHOR]

Boron stress is an environmental factor affecting plant development and production. Recently, microRNAs (miRNAs) have been found to be involved in several plant processes such as growth regulation and stress responses. In this study, miRNAs associated with boron stress were identified and characterized in barley. miRNA profiles were also comparatively analyzed between root and leave samples. A total of 31 known and 3 new miRNAs were identified in barley; 25 of them were found to respond to boron treatment. Several miRNAs were expressed in a tissue specific manner; for example, miR156d, miR171a, miR397, and miR444a were only detected in leaves. Additionally, a total of 934 barley transcripts were found to be specifically targeted and degraded by miRNAs. In silico analysis of miRNA target genes demonstrated that many miRNA targets are conserved transcription factors such as Squamosa promoter-binding protein, Auxin response factor (ARF), and the MYB transcription factor family. A majority of these targets were responsible for plant growth and response to environmental changes. We also propose that some of the miRNAs in barley such as miRNA408 might play critical roles against boron exposure. In conclusion, barley may use several pathways and cellular processes targeted by miRNAs to cope with boron stress. [ABSTRACT FROM AUTHOR]

A total of 28,432 unique contigs (25,371 in consensus contigs and 3,061 as singletons) were assembled from all 268,786 cotton ESTs currently available. Several in silico approaches [comparative genomics, Blast, Gene Ontology (GO) analysis, and pathway enrichment by Kyoto Encyclopedia of Genes and Genomes (KEGG)] were employed to investigate global functions of the cotton transcriptome. Cotton EST contigs were clustered into 5,461 groups with a maximum cluster size of 196 members. A total of 27,956 indel mutants and 149,616 single nucleotide polymorphisms (SNPs) were identified from consensus contigs. Interestingly, many contigs with significantly high frequencies of indels or SNPs encode transcription factors and protein kinases. In a comparison with six model plant species, cotton ESTs show the highest overall similarity to grape. A total of 87 cotton miRNAs were identified; 59 of these have not been reported previously from experimental or bioinformatics investigations. We also predicted 3,260 genes as miRNAs targets, which are associated with multiple biological functions, including stress response, metabolism, hormone signal transduction and fiber development. We identified 151 and 4,214 EST-simple sequence repeats (SSRs) from contigs and raw ESTs respectively. To make these data widely available, and to facilitate access to EST-related genetic information, we integrated our results into a comprehensive, fully downloadable web-based cotton EST database (www.leonxie.com). [ABSTRACT FROM AUTHOR]

14-3-3s are a class of conserved proteins in eukaryotes involved in almost all cellular processes. Published studies have shown that this family plays a role in response to stress conditions. In this study, comparative genomics identified thirty-one 14-3-3 cDNAs encoding 25 unique proteins with transcripts that were sufficiently divergent in the coding regions to define different 14-3-3 members. Phylogenetic analysis indicated that the cotton 14-3-3 family was similar to the family in the plant model species Arabidopsis and rice. The multiple copies of 14-3-3 genes in cotton are suggested to be the result of a recent duplication event after the divergence of cotton from its progenitor species. 14-3-3s are differentially expressed in a range of cotton organs as well as under different environmental conditions. Salinity and drought stress significantly induced the altered expression of 14-3-3 genes, suggesting that 14-3-3s play a role in cotton response to environmental abiotic stress. The 14-3-3 gene CGF14-4 was more sensitive to both drought and salinity stress with a 10.2-fold change under 1% of PEG treatment, while other 14-3-3s responded only to either drought or salinity stress. These results implied that specific isoforms of 14-3-3s play different regulatory roles in stress response besides their role in development. 14-3-3s were more sensitive to abiotic stress in roots than in leaves, suggesting that the root is more sensitive to stress treatment than leaves. The differential expression pattern of 14-3-3s also suggests the occurrence of functional divergence among specific isoforms. [ABSTRACT FROM AUTHOR]

Auxin response factors (ARFs) are a family of transcription factors that play an important role of auxin regulation through their binding with auxin response elements. ARF genes are represented by a large multigene family in plants; however, to our knowledge, the ARF gene family has not been well studied and characterized in sweet potatoes. In this study, a total of 25 ARF genes were identified in Ipomea trifida. The identified ItrARF genes' conserved motifs, chromosomal locations, phylogenetic relationships, and their protein characteristics were systemically investigated using different bioinformatics tools. The expression patterns of ItfARF genes were analyzed within the storage roots and normal roots at an early stage of development. ItfARF16b and ItfARF16c were both highly expressed in the storage root, with minimal to no expression in the normal root. ItfARF6a and ItfARF10a exhibited higher expression in the normal root but not in the storage root. Subsequently, ItfARF1a, ItfARF2b, ItfARF3a, ItfARF6b, ItfARF8a, ItfARF8b, and ItfARF10b were expressed in both root types with moderate to high expression for each. All ten of these ARF genes and their prominent expression signify their importance within the development of each respective root type. This study provides comprehensive information regarding the ARF family in sweet potatoes, which will be useful for future research to discover further functional verification of these ItfARF genes. [ABSTRACT FROM AUTHOR]

Cassava, a crucial tropical crop, faces challenges from cold stress, necessitating an exploration of its molecular response. Here, we investigated the role of DNA methylation in moderating the response to moderate cold stress (10 °C) in cassava. Using whole-genome bisulfite sequencing, we examined DNA methylation patterns in leaf blades and petioles under control conditions, 5 h, and 48 h of cold stress. Tissue-specific responses were observed, with leaf blades exhibiting subtle changes, while petioles displayed a pronounced decrease in methylation levels under cold stress. We identified cold stress-induced differentially methylated regions (DMRs) that demonstrated both tissue and treatment specificity. Importantly, these DMRs were enriched in genes with altered expression, implying functional relevance. The cold-response transcription factor ERF105 associated with DMRs emerged as a significant and conserved regulator across tissues and treatments. Furthermore, we investigated DNA methylation dynamics in transposable elements, emphasizing the sensitivity of MITEs with bHLH binding motifs to cold stress. These findings provide insights into the epigenetic regulation of response to cold stress in cassava, contributing to an understanding of the molecular mechanisms underlying stress adaptation in this tropical plant. • Cassava displayed tissue-specific DNA methylation responses to cold stress. • Integration of DNA methylation and gene expression data revealed intricate correlations. • The transcription factor ERF105 is a significant and conserved regulator across tissues and treatments under cold stress. • Transposable elements, particularly MITEs with bHLH binding motifs, showed sensitivity to cold stress. [ABSTRACT FROM AUTHOR]

Abstract: MicroRNAs are a recently identified class of small regulatory RNAs that target more than 30% protein-coding genes. Elevating evidence shows that miRNAs play a critical role in many biological processes, including developmental timing, tissue differentiation, and response to chemical exposure. In this study, we applied a computational approach to analyze expressed sequence tags, and identified 32 miRNAs belonging to 22 miRNA families, in three earthworm species Eisenia fetida, Eisenia andrei, and Lumbricus rubellus. These newly identified earthworm miRNAs possess a difference of 2–4 nucleotides from their homologous counterparts in Caenorhabditis elegans. They also share similar features with other known animal miRNAs, for instance, the nucleotide U being dominant in both mature and pre-miRNA sequences, particularly in the first position of mature miRNA sequences at the 5′ end. The newly identified earthworm miRNAs putatively regulate mRNA genes that are involved in many important biological processes and pathways related to development, growth, locomotion, and reproduction as well as response to stresses, particularly oxidative stress. Future efforts will focus on experimental validation of their presence and target mRNA genes to further elucidate their biological functions in earthworms. [ABSTRACT FROM AUTHOR]