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.

AUXIN RESPONSE FACTOR7 integrates gibberellin and auxin signaling via interactions between DELLA and AUX/IAA proteins to regulate cambial activity in poplar.

Cambial development in the stems of perennial woody species is rigorously regulated by phytohormones. Auxin and gibberellin (GA) play crucial roles in stimulating cambial activity in poplar (Populus spp.). In this study, we show that the DELLA protein REPRESSOR of ga1-3 Like 1 (RGL1), AUXIN RESPONSE FACTOR 7 (ARF7), and Aux/INDOLE-3-ACETIC ACID 9 (IAA9) form a ternary complex that mediates crosstalk between the auxin and GA signaling pathways in poplar stems during cambial development. Biochemical analysis revealed that ARF7 physically interacts with RGL1 and IAA9 through distinct domains. The arf7 loss-of-function mutant showed markedly attenuated responses to auxin and GA, whereas transgenic poplar plants overexpressing ARF7 displayed strongly improved cambial activity. ARF7 directly binds to the promoter region of the cambial stem cell regulator WOX4 to modulate its expression, thus integrating auxin and GA signaling to regulate cambial activity. Furthermore, the direct activation of PIN-FORMED 1 expression by ARF7 in the RGL1-ARF7-IAA9 module increased GA-dependent cambial activity via polar auxin transport. Collectively, these findings reveal that the crosstalk between auxin and GA signaling mediated by the RGL1-ARF7-IAA9 module is crucial for the precise regulation of cambial development in poplar.

A systematic analysis of the phloem protein 2 (PP2) proteins in Gossypium hirsutum reveals that GhPP2-33 regulates salt tolerance.

BACKGROUND: Phloem protein 2 (PP2) proteins play a vital role in the Phloem-based defense (PBD) and participate in many abiotic and biotic stress. However, research on PP2 proteins in cotton is still lacking. RESULTS: A total of 25, 23, 43, and 47 PP2 genes were comprehensively identified and characterized in G.arboretum, G.raimondii, G.barbadense, and G.hirsutum. The whole genome duplication (WGD) and allopolyploidization events play essential roles in the expansion of PP2 genes. The promoter regions of GhPP2 genes contain many cis-acting elements related to abiotic stress and the weighted gene co-expression network analysis (WGCNA) analysis displayed that GhPP2s could be related to salt stress. The qRT-PCR assays further confirmed that GhPP2-33 could be dramatically upregulated during the salt treatment. And the virus-induced gene silencing (VIGS) experiment proved that the silencing of GhPP2-33 could decrease salt tolerance. CONCLUSIONS: The results in this study not only offer new perspectives for understanding the evolution of PP2 genes in cotton but also further explore their function under salt stress.

Genome-wide analysis of SET domain genes and the function of GhSDG51 during salt stress in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Cotton, being extensively cultivated, holds immense economic significance as one of the most prominent crops globally. The SET (Su(var), E, and Trithorax) domain-containing protein is of significant importance in plant development, growth, and response to abiotic stress by modifying the lysine methylation status of histone. However, the comprehensive identification of SET domain genes (SDG) have not been conducted in upland cotton (Gossypium hirsutum L.). RESULTS: A total of 229 SDGs were identified in four Gossypium species, including G. arboretum, G. raimondii, G. hirsutum, and G. barbadense. These genes could distinctly be divided into eight groups. The analysis of gene structure and protein motif revealed a high degree of conservation among the SDGs within the same group. Collinearity analysis suggested that the SDGs of Gossypium species and most of the other selected plants were mainly expanded by dispersed duplication events and whole genome duplication (WGD) events. The allopolyploidization event also has a significant impact on the expansion of SDGs in tetraploid Gossypium species. Furthermore, the characteristics of these genes have been relatively conserved during the evolution. Cis-element analysis revealed that GhSDGs play a role in resistance to abiotic stresses and growth development. Furthermore, the qRT-PCR results have indicated the ability of GhSDGs to respond to salt stress. Co-expression analysis revealed that GhSDG51 might co-express with genes associated with salt stress. In addition, the silencing of GhSDG51 in cotton by the virus-induced gene silencing (VIGS) method suggested a potential positive regulatory role of GhSDG51 in salt stress. CONCLUSIONS: The results of this study comprehensively analyze the SDGs in cotton and provide a basis for understanding the biological role of SDGs in the stress resistance in upland cotton.

Public Surveillance of Social Media for Suicide Using Advanced Deep Learning Models in Japan: Time Series Study From 2012 to 2022.

BACKGROUND: Social media platforms have been increasingly used to express suicidal thoughts, feelings, and acts, raising public concerns over time. A large body of literature has explored the suicide risks identified by people's expressions on social media. However, there is not enough evidence to conclude that social media provides public surveillance for suicide without aligning suicide risks detected on social media with actual suicidal behaviors. Corroborating this alignment is a crucial foundation for suicide prevention and intervention through social media and for estimating and predicting suicide in countries with no reliable suicide statistics. OBJECTIVE: This study aimed to corroborate whether the suicide risks identified on social media align with actual suicidal behaviors. This aim was achieved by tracking suicide risks detected by 62 million tweets posted in Japan over a 10-year period and assessing the locational and temporal alignment of such suicide risks with actual suicide behaviors recorded in national suicide statistics. METHODS: This study used a human-in-the-loop approach to identify suicide-risk tweets posted in Japan from January 2013 to December 2022. This approach involved keyword-filtered data mining, data scanning by human efforts, and data refinement via an advanced natural language processing model termed Bidirectional Encoder Representations from Transformers. The tweet-identified suicide risks were then compared with actual suicide records in both temporal and spatial dimensions to validate if they were statistically correlated. RESULTS: Twitter-identified suicide risks and actual suicide records were temporally correlated by month in the 10 years from 2013 to 2022 (correlation coefficient=0.533; P<.001); this correlation coefficient is higher at 0.652 when we advanced the Twitter-identified suicide risks 1 month earlier to compare with the actual suicide records. These 2 indicators were also spatially correlated by city with a correlation coefficient of 0.699 (P<.001) for the 10-year period. Among the 267 cities with the top quintile of suicide risks identified from both tweets and actual suicide records, 73.5% (n=196) of cities overlapped. In addition, Twitter-identified suicide risks were at a relatively lower level after midnight compared to a higher level in the afternoon, as well as a higher level on Sundays and Saturdays compared to weekdays. CONCLUSIONS: Social media platforms provide an anonymous space where people express their suicidal thoughts, ideation, and acts. Such expressions can serve as an alternative source to estimating and predicting suicide in countries without reliable suicide statistics. It can also provide real-time tracking of suicide risks, serving as an early warning for suicide. The identification of areas where suicide risks are highly concentrated is crucial for location-based mental health planning, enabling suicide prevention and intervention through social media in a spatially and temporally explicit manner.

Comparison of endoscopic bilateral areolar and robotic-assisted bilateral axillo-breast approach thyroidectomy in differentiated thyroid carcinoma: a propensity-matched retrospective cohort study.

BACKGROUND: Robot-assisted and endoscopic thyroidectomy are superior to conventional open thyroidectomy in improving cosmetic outcomes and postoperative quality of life. The procedure of these thyroidectomies was similar in terms of surgical view, feasibility, and invasiveness. However, it remains uncertain whether the robotic-assisted bilateral axilla-breast approach (BABA) was superior to the endoscopic bilateral areolar approach (BAA) thyroidectomy. This study aimed to investigate the clinical benefit of these two surgical procedures to evaluate the difference between these two surgical procedures by comparing the pathological and surgical outcomes of endoscopic BAA and robotic-assisted BABA thyroidectomy in differentiated thyroid carcinoma. METHODS: From November 2018 to September 2021, 278 patients with differentiated thyroid carcinoma underwent BABA robot-assisted, and 49 underwent BAA approach endoscopic thyroidectomy. Of these patients, we analyzed 42 and 135 patients of endoscopic and robotic matched pairs using 1:4 propensity score matching and retrospective cohort study methods. These two groups were retrospectively compared by surgical outcomes, clinicopathological characteristics, and postoperative complications. RESULTS: The mean operation time was significantly longer in the EG than in the RG (p < 0.001), The number of retrieved lymph nodes was significantly lower in the ET group than in the RT group (p < 0.001). The mean maximum diameter of the thyroid was more expansive in the EG than in the RG (p = 0.04). There were no significant differences in the total drainage amount and drain insertion days between the two groups (p = 0.241, p = 0.316, respectively). Both groups showed that cosmetic satisfaction (p = 0.837) and pain score (p = 0.077) were similar. There were no significant differences in complication frequencies. CONCLUSION: Robotic and endoscopic thyroidectomy are similar minimally invasive thyroid surgeries, each with its advantages, both of which can achieve the expected surgical outcomes. TRIAL REGISTRATION: Retrospectively registered.

The CCCH-Type Zinc-Finger Protein GhC3H20 Enhances Salt Stress Tolerance in Arabidopsis thaliana and Cotton through ABA Signal Transduction Pathway.

The CCCH zinc-finger protein contains a typical C3H-type motif widely existing in plants, and it plays an important role in plant growth, development, and stress responses. In this study, a CCCH zinc-finger gene, GhC3H20, was isolated and thoroughly characterized to regulate salt stress in cotton and Arabidopsis. The expression of GhC3H20 was up-regulated under salt, drought, and ABA treatments. GUS activity was detected in the root, stem, leaves, and flowers of ProGhC3H20::GUS transgenic Arabidopsis. Compared with the control, the GUS activity of ProGhC3H20::GUS transgenic Arabidopsis seedlings under NaCl treatment was stronger. Through the genetic transformation of Arabidopsis, three transgenic lines of 35S-GhC3H20 were obtained. Under NaCl and mannitol treatments, the roots of the transgenic lines were significantly longer than those of the wild-type (WT) Arabidopsis. The leaves of the WT turned yellow and wilted under high-concentration salt treatment at the seedling stage, while the leaves of the transgenic Arabidopsis lines did not. Further investigation showed that compared with the WT, the content of catalase (CAT) in the leaves of the transgenic lines was significantly higher. Therefore, compared with the WT, overexpression of GhC3H20 enhanced the salt stress tolerance of transgenic Arabidopsis. A virus-induced gene silencing (VIGS) experiment showed that compared with the control, the leaves of pYL156-GhC3H20 plants were wilted and dehydrated. The content of chlorophyll in pYL156-GhC3H20 leaves was significantly lower than those of the control. Therefore, silencing of GhC3H20 reduced salt stress tolerance in cotton. Two interacting proteins (GhPP2CA and GhHAB1) of GhC3H20 have been identified through a yeast two-hybrid assay. The expression levels of PP2CA and HAB1 in transgenic Arabidopsis were higher than those in the WT, and pYL156-GhC3H20 had expression levels lower than those in the control. GhPP2CA and GhHAB1 are the key genes involved in the ABA signaling pathway. Taken together, our findings demonstrate that GhC3H20 may interact with GhPP2CA and GhHAB1 to participate in the ABA signaling pathway to enhance salt stress tolerance in cotton.

Non-functional GoFLA19s are responsible for the male sterility caused by hybrid breakdown in cotton (Gossypium spp.).

Hybrid breakdown (HB) functions as a common reproductive barrier and reduces hybrid fitness in many species, including cotton. However, the related genes and the underlying genetic mechanisms of HB in cotton remain unknown. Here, we found that the photosensitive genetic male sterile line CCRI9106 was a hybrid progeny of Gossypium hirsutum and Gossypium barbadense and probably a product of HB. Fine mapping with F2 s (CCRI9106 × G. hirsutum/G. barbadense lines) identified a pair of male sterility genes GoFLA19s (encoding fasciclin-like arabinogalactan family protein) located on chromosomes A12 and D12. Crucial variations occurring in the fasciclin-like domain and the arabinogalactan protein domain were predicted to cause the non-functionalization of GbFLA19-D and GhFLA19-A. CRISPR/Cas9-mediated knockout assay confirmed the effects of GhFLA19s on male sterility. Sequence alignment analyses showed that variations in GbFLA19-D and GhFLA19-A likely occurred after the formation of allotetraploid cotton species. GoFLA19s are specifically expressed in anthers and contribute to tapetal development, exine assembly, intine formation, and pollen grain maturation. RNA-sequencing and quantitative reverse transcriptase-polymerase chain reaction analyses illustrated that genes related to these biological processes were significantly downregulated in the mutant. Our research on male sterility genes, GoFLA19s, improves the understanding of the molecular characteristics and evolutionary significance of HB in interspecific hybrid breeding.

Systematic analysis of CNGCs in cotton and the positive role of GhCNGC32 and GhCNGC35 in salt tolerance.

BACKGROUND: Cyclic nucleotide-gated ion channels (CNGCs) are calcium-permeable channels that participate in a variety of biological functions, such as signaling pathways, plant development, and environmental stress and stimulus responses. Nevertheless, there have been few studies on CNGC gene family in cotton. RESULTS: In this study, a total of 114 CNGC genes were identified from the genomes of 4 cotton species. These genes clustered into 5 main groups: I, II, III, IVa, and IVb. Gene structure and protein motif analysis showed that CNGCs on the same branch were highly conserved. In addition, collinearity analysis showed that the CNGC gene family had expanded mainly by whole-genome duplication (WGD). Promoter analysis of the GhCNGCs showed that there were a large number of cis-acting elements related to abscisic acid (ABA). Combination of transcriptome data and the results of quantitative RT-PCR (qRT-PCR) analysis revealed that some GhCNGC genes were induced in response to salt and drought stress and to exogenous ABA. Virus-induced gene silencing (VIGS) experiments showed that the silencing of the GhCNGC32 and GhCNGC35 genes decreased the salt tolerance of cotton plants (TRV:00). Specifically, physiological indexes showed that the malondialdehyde (MDA) content in gene-silenced plants (TRV:GhCNGC32 and TRV:GhCNGC35) increased significantly under salt stress but that the peroxidase (POD) activity decreased. After salt stress, the expression level of ABA-related genes increased significantly, indicating that salt stress can trigger the ABA signal regulatory mechanism. CONCLUSIONS: we comprehensively analyzed CNGC genes in four cotton species, and found that GhCNGC32 and GhCNGC35 genes play an important role in cotton salt tolerance. These results laid a foundation for the subsequent study of the involvement of cotton CNGC genes in salt tolerance.

A Comprehensive Gene Co-Expression Network Analysis Reveals a Role of GhWRKY46 in Responding to Drought and Salt Stresses.

Abiotic stress, such as drought and salinity stress, seriously inhibit the growth and development of plants. Therefore, it is vital to understand the drought and salinity resistance mechanisms to enable cotton to provide more production under drought and salt conditions. In this study, we identified 8806 and 9108 differentially expressed genes (DEGs) through a comprehensive analysis of transcriptomic data related to the PEG-induced osmotic and salt stress in cotton. By performing weighted gene co-expression network analysis (WGCNA), we identified four co-expression modules in PEG treatment and five co-expression modules in salinity stress, which included 346 and 324 predicted transcription factors (TFs) in these modules, respectively. Correspondingly, whole genome duplication (WGD) events mainly contribute to the expansion of those TFs. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analyses revealed those different modules were associated with stress resistance, including regulating macromolecule metabolic process, peptidase activity, transporter activity, lipid metabolic process, and responses to stimulus. Quantitative RT-PCR analysis was used to confirm the expression levels of 15 hub TFs in PEG6000 and salinity treatments. We found that the hub gene GhWRKY46 could alter salt and PEG-induced drought resistance in cotton through the virus-induced gene silencing (VIGS) method. Our results provide a preliminary framework for further investigation of the cotton response to salt and drought stress, which is significant to breeding salt- and drought-tolerant cotton varieties.

A Comprehensive Identification and Function Analysis of Serine/Arginine-Rich (SR) Proteins in Cotton (Gossypium spp.).

As one of the most important factors in alternative splicing (AS) events, serine/arginine-rich (SR) proteins not only participate in the growth and development of plants but also play pivotal roles in abiotic stresses. However, the research about SR proteins in cotton is still lacking. In this study, we performed an extensive comparative analysis of SR proteins and determined their phylogeny in the plant lineage. A total of 169 SR family members were identified from four Gossypium species, and these genes could be divided into eight distinct subfamilies. The domain, motif distribution and gene structure of cotton SR proteins are conserved within each subfamily. The expansion of SR genes is mainly contributed by WGD and allopolyploidization events in cotton. The selection pressure analysis showed that all the paralogous gene pairs were under purifying selection pressure. Many cis-elements responding to abiotic stress and phytohormones were identified in the upstream sequences of the GhSR genes. Expression profiling suggested that some GhSR genes may involve in the pathways of plant resistance to abiotic stresses. The WGCNA analysis showed that GhSCL-8 co-expressed with many abiotic responding related genes in a salt-responding network. The Y2H assays showed that GhSCL-8 could interact with GhSRs in other subfamilies. The subcellular location analysis showed that GhSCL-8 is expressed in the nucleus. The further VIGS assays showed that the silencing of GhSCL-8 could decrease salt tolerance in cotton. These results expand our knowledge of the evolution of the SR gene family in plants, and they will also contribute to the elucidation of the biological functions of SR genes in the future.

A Comprehensive Analysis of the DUF4228 Gene Family in Gossypium Reveals the Role of GhDUF4228-67 in Salt Tolerance.

Soil salinization conditions seriously restrict cotton yield and quality. Related studies have shown that the DUF4228 proteins are pivotal in plant resistance to abiotic stress. However, there has been no systematic identification and analysis of the DUF4228 gene family in cotton and their role in abiotic stress. In this study, a total of 308 DUF4228 genes were identified in four Gossypium species, which were divided into five subfamilies. Gene structure and protein motifs analysis showed that the GhDUF4228 proteins were conserved in each subfamily. In addition, whole genome duplication (WGD) events and allopolyploidization might play an essential role in the expansion of the DUF4228 genes. Besides, many stress-responsive (MYB, MYC) and hormone-responsive (ABA, MeJA) related cis-elements were detected in the promoters of the DUF4228 genes. The qRT-PCR results showed that GhDUF4228 genes might be involved in the response to abiotic stress. VIGS assays and the measurement of relative water content (RWC), Proline content, POD activity, and malondialdehyde (MDA) content indicated that GhDUF4228-67 might be a positive regulator of cotton response to salt stress. The results in this study systematically characterized the DUF4228s in Gossypium species and will provide helpful information to further research the role of DUF4228s in salt tolerance.

Comprehensive identification and expression analysis of B-Box genes in cotton.

BACKGROUND: B-BOX (BBX) proteins are zinc-finger transcription factors with one or two BBX domains and sometimes a CCT domain. These proteins play an essential role in regulating plant growth and development, as well as in resisting abiotic stress. So far, the BBX gene family has been widely studied in other crops. However, no one has systematically studied the BBX gene in cotton. RESULTS: In the present study, 17, 18, 37 and 33 BBX genes were detected in Gossypium arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively, via genome-wide identification. Phylogenetic analysis showed that all BBX genes were divided into 5 main categories. The protein motifs and exon/intron structures showed that each group of BBX genes was highly conserved. Collinearity analysis revealed that the amplification of BBX gene family in Gossypium spp. was mainly through segmental replication. Nonsynonymous (Ka)/ synonymous (Ks) substitution ratios indicated that the BBX gene family had undergone purification selection throughout the long-term natural selection process. Moreover, transcriptomic data showed that some GhBBX genes were highly expressed in floral organs. The qRT-PCR results showed that there were significant differences in GhBBX genes in leaves and shoot apexes between early-maturing materials and late-maturing materials at most periods. Yeast two-hybrid results showed that GhBBX5/GhBBX23 and GhBBX8/GhBBX26 might interact with GhFT. Transcriptome data analysis and qRT-PCR verification showed that different GhBBX genes had different biological functions in abiotic stress and phytohormone response. CONCLUSIONS: Our comprehensive analysis of BBX in G. hirsutum provided a basis for further study on the molecular role of GhBBXs in regulating flowering and cotton resistance to abiotic stress.

Cytokinin signaling localized in phloem noncell-autonomously regulates cambial activity during secondary growth of Populus stems.

The regulation of cytokinin on secondary vascular development has been uncovered by modulating cytokinin content. However, it remains unclear how cytokinin enriched in developing secondary phloem regulates cambium activity in poplar. Here, we visualized the gradient distribution of cytokinin with a peak in the secondary phloem of poplar stem via immunohistochemical imaging, and determined the role of phloem-located cytokinin signaling during wood formation. We generated transgenic poplar harboring cytokinin oxidase/dehydrogenase (CKX)2, a gene encoding a cytokinin degrading enzyme, driven by the phloem-specific CLE41b promoter, indicating that the disruption of the cytokinin gradient pattern restricts the cambial activity. The RNA interference-based knockdown of the histidine kinase (HK) genes encoding cytokinin receptors specifically in secondary phloem significantly compromised the division activity of cambial cells, whereas the phloem-specific expression of a type-B response regulator (RR) transcription factor stimulated cambial proliferation, providing evidence for the noncell-autonomous regulation of local cytokinin signaling on the cambial activity. Moreover, the cambium-specific knockdown of HKs also led to restricted cambial activity, and the defects were aggravated by the reduced cytokinin accumulation. Our results showed that local cytokinin signaling in secondary phloem regulates cambial activity noncell-autonomously, and coordinately with its local signaling in cambium.

The microRNA476a-RFL module regulates adventitious root formation through a mitochondria-dependent pathway in Populus.

For woody plants, clonal propagation efficiency is largely determined by adventitious root (AR) formation at the bases of stem cuttings. However, our understanding of the molecular mechanisms contributing to AR morphogenesis in trees remains limited, despite the importance of vegetative propagation, currently the most common practice for tree breeding and commercialization. Here, we identified Populus-specific miR476a as a regulator of wound-induced adventitious rooting that acts by orchestrating mitochondrial homeostasis. MiR476a exhibited inducible expression during AR formation and directly targeted several Restorer of Fertility like (RFL) genes encoding mitochondrion-localized pentatricopeptide repeat proteins. Genetic modification of miR476a-RFL expression revealed that miR476a/RFL-mediated dynamic regulation of mitochondrial homeostasis influences AR formation in poplar. Mitochondrial perturbation via exogenous application of a chemical inhibitor indicated that miR476a/RFL-directed AR formation depends on mitochondrial regulation that acts via auxin signaling. Our results thus establish a microRNA-directed mitochondrion-auxin signaling cascade required for AR development, providing insights into the role of mitochondrial regulation in the developmental plasticity of plants.

Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic Arabidopsis.

Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.

Deficiencies in the formation and regulation of anther cuticle and tryphine contribute to male sterility in cotton PGMS line.

BACKGROUND: Male sterility is a simple and efficient pollination control system that is widely exploited in hybrid breeding. In upland cotton, CCRI9106, a photosensitive genetic male sterile (PGMS) mutant isolated from CCRI040029, was reported of great advantages to cotton heterosis. However, little information concerning the male sterility of CCRI9106 is known. Here, comparative transcriptome analysis of CCRI9106 (the mutant, MT) and CCRI040029 (the wild type, WT) anthers in Anyang (long-day, male sterile condition to CCRI9106) was performed to reveal the potential male sterile mechanism of CCRI9106. RESULTS: Light and electron microscopy revealed that the male sterility phenotype of MT was mainly attributed to irregularly exine, lacking tryphine and immature anther cuticle. Based on the cytological characteristics of MT anthers, anther RNA libraries (18 in total) of tetrad (TTP), late uninucleate (lUNP) and binucleate (BNP) stages in MT and WT were constructed for transcriptomic analysis, therefore revealing a total of 870,4 differentially expressed genes (DEGs). By performing gene expression pattern analysis and protein-protein interaction (PPI) networks construction, we found down-regulation of DEGs, which enriched by the lipid biosynthetic process and the synthesis pathways of several types of secondary metabolites such as terpenoids, flavonoids and steroids, may crucial to the male sterility phenotype of MT, and resulting in the defects of anther cuticle and tryphine, even the irregularly exine. Furthermore, several lipid-related genes together with ABA-related genes and MYB transcription factors were identified as hub genes via weighted gene co-expression network analysis (WGCNA). Additionally, the ABA content of MT anthers was reduced across all stages when compared with WT anthers. At last, genes related to the formation of anther cuticle and tryphine could activated in MT under short-day condition. CONCLUSIONS: We propose that the down-regulation of genes related to the assembly of anther cuticle and tryphine may lead to the male sterile phenotype of MT, and MYB transcription factors together with ABA played key regulatory roles in these processes. The conversion of fertility in different photoperiods may closely relate to the functional expression of these genes. These findings contribute to elucidate the mechanism of male sterility in upland 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.

Auxin-mediated Aux/IAA-ARF-HB signaling cascade regulates secondary xylem development in Populus.

Wood development is strictly regulated by various phytohormones and auxin plays a central regulatory role in this process. However, how the auxin signaling is transducted in developing secondary xylem during wood formation in tree species remains unclear. Here, we identified an Aux/INDOLE-3-ACETIC ACID 9 (IAA9)-AUXIN RESPONSE FACTOR 5 (ARF5) module in Populus tomentosa as a key mediator of auxin signaling to control early developing xylem development. PtoIAA9, a canonical Aux/IAA gene, is predominantly expressed in vascular cambium and developing secondary xylem and induced by exogenous auxin. Overexpression of PtoIAA9m encoding a stabilized IAA9 protein significantly represses secondary xylem development in transgenic poplar. We further showed that PtoIAA9 interacts with PtoARF5 homologs via the C-terminal III/IV domains. The truncated PtoARF5.1 protein without the III/IV domains rescued defective phenotypes caused by PtoIAA9m. Expression analysis showed that the PtoIAA9-PtoARF5 module regulated the expression of genes associated with secondary vascular development in PtoIAA9m- and PtoARF5.1-overexpressing plants. Furthermore, PtoARF5.1 could bind to the promoters of two Class III homeodomain-leucine zipper (HD-ZIP III) genes, PtoHB7 and PtoHB8, to modulate secondary xylem formation. Taken together, our results suggest that the Aux/IAA9-ARF5 module is required for auxin signaling to regulate wood formation via orchestrating the expression of HD-ZIP III transcription factors in poplar.

The MicroRNA390/TRANS-ACTING SHORT INTERFERING RNA3 Module Mediates Lateral Root Growth under Salt Stress via the Auxin Pathway.

Salt-induced developmental plasticity in a plant root system strongly depends on auxin signaling. However, the molecular events underlying this process are poorly understood. MicroRNA390 (miR390), trans-actin small interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORs (ARFs) form a regulatory module involved in controlling lateral root (LR) growth. Here, we found that miR390 expression was strongly induced by exposure to salt during LR formation in poplar (Populus spp.) plants. miR390 overexpression stimulated LR development and increased salt tolerance, whereas miR390 knockdown caused by a short tandem target mimic repressed LR growth and compromised salt resistance. ARF3.1, ARF3.2, and ARF4 expression was inhibited significantly by the presence of salt, and transcript abundance was decreased dramatically in the miR390-overexpressing line but increased in the miR390-knockdown line. Constitutive expression of ARF4m harboring mutated trans-acting small interfering ARF-binding sites removed the salt resistance of the miR390 overexpressors. miR390 positively regulated auxin signaling in LRs subjected to salt, but ARF4 inhibited auxin signaling. Salinity stabilized the poplar Aux/IAA repressor INDOLE-3-ACETIC ACID17.1, and overexpression of an auxin/salt-resistant form of this repressor suppressed LR growth in miR390-overexpressing and ARF4-RNA interfering lines in the presence of salt. Thus, the miR390/TAS3/ARFs module is a key regulator, via modulating the auxin pathway, of LR growth in poplar subjected to salt stress.