The Verticillium dahliae Effector VdPHB1 Promotes Pathogenicity in Cotton and Interacts with the Immune Protein GhMC4.

Verticillium dahliae is a kind of pathogenic fungus that brings about wilt disease and great losses in cotton. The molecular mechanism of the effectors in V. dahliae regulating cotton immunity remains largely unknown. Here, we identified an effector of V. dahliae, VdPHB1, whose gene expression is highly induced by infection. The VdPHB1 protein is localized to the intercellular space of cotton plants. Knock-out of the VdPHB1 gene in V. dahliae had no effect on pathogen growth, but decreased the virulence in cotton. VdPHB1 ectopically expressed Arabidopsis plants were growth-inhibited and significantly susceptible to V. dahliae. Further, VdPHB1 interacted with the type II metacaspase GhMC4. GhMC4 gene-silenced cotton plants were more sensitive to V. dahliae with reduced expression of pathogen defense-related and programmed cell death genes. The accumulation of GhMC4 protein was concurrently repressed when VdPHB1 protein was expressed during infection. In summary, these results have revealed a novel molecular mechanism of virulence regulation that the secreted effector VdPHB1 represses the activity of cysteine protease for helping V. dahliae infection in cotton.

Transcriptional regulation of phospholipid transport in cotton fiber elongation by GhMYB30D04-GhHD1 interaction complex.

Cotton fiber length is basically determined by well-coordinated gene expression and phosphatidylinositol phosphates (PIPs) accumulation during fiber elongation but the regulatory mechanism governing PIPs transport remains unknown. Here, we report a MYB transcription factor GhMYB30D04 in Gossypium hirsutum that promotes fiber elongation through modulating the expression of PIP transporter gene GhLTPG1. Knockout of GhMYB30D04 gene in cotton (KO) results in a reduction of GhLTPG1 transcripts with lower accumulation of PIPs, leading to shorter fibers and lower fiber yield. Conversely, GhMYB30D04 overexpression (GhMYB30D04-OE) causes richer PIPs and longer cotton fibers, mimicking the effects of exogenously applying PIPs on the ovules of GhMYB30D04-KO and wild type. Furthermore, GhMYB30D04 interacts with GhHD1, the crucial transcription factor of fiber initiation, to form an activation complex stabilized by PIPs, both of which upregulate GhLTPG1 expression. Comparative omics-analysis revealed that higher and extended expressions of LTPG1 in fiber elongation mainly correlate with the variations of the GhMYB30D04 gene between two cotton allotetraploids, contributing to longer fiber in G. babardense. Our work clarifies a mechanism by which GhHD1-GhMYB30D04 form a regulatory module of fiber elongation to tightly control PIP accumulation. Our work still has an implication that GhMYB30D04-GhHD1 associates with development transition from fiber initiation to elongation.

GhXB38D represses cotton fibre elongation through ubiquitination of ethylene biosynthesis enzymes GhACS4 and GhACO1.

Ethylene plays an essential role in the development of cotton fibres. Ethylene biosynthesis in plants is elaborately regulated by the activities of key enzymes, 1-aminocyclopropane-1-carboxylate oxidase (ACO) and 1-aminocyclopropane-1-carboxylate synthase (ACS); however, the potential mechanism of post-translational modification of ACO and ACS to control ethylene synthesis in cotton fibres remains unclear. Here, we identify an E3 ubiquitin ligase, GhXB38D, that regulates ethylene biosynthesis during fibre elongation in cotton. GhXB38D gene is highly expressed in cotton fibres during the rapid elongation stage. Suppressing GhXB38D expression in cotton significantly enhanced fibre elongation and length, accompanied by the up-regulation of genes associated with ethylene signalling and fibre elongation. We demonstrated that GhXB38D interacts with the ethylene biosynthesis enzymes GhACS4 and GhACO1 in elongating fibres and specifically mediates their ubiquitination and degradation. The inhibition of GhXB38D gene expression increased the stability of GhACS4 and GhACO1 proteins in cotton fibres and ovules, resulting in an elevated concentration of ethylene. Our findings highlight the role of GhXB38D as a regulator of ethylene synthesis by ubiquitinating ACS4 and ACO1 proteins and modulating their stability. GhXB38D acts as a negative regulator of fibre elongation and serves as a potential target for enhancing cotton fibre yield and quality through gene editing strategy.

Arabidopsis calcium-dependent protein kinase CPK6 regulates drought tolerance under high nitrogen by the phosphorylation of NRT1.1.

Nitrogen (N) is an essential macronutrient for plant growth and development, and its availability is regulated to some extent by drought stress. Calcium-dependent protein kinases (CPKs) are a unique family of Ca2+ sensors with diverse functions in N uptake and drought-tolerance signaling pathways; however, how CPKs are involved in the crosstalk between drought stress and N transportation remains largely unknown. Here, we identify the drought-tolerance function of Arabidopsis CPK6 under high N conditions. CPK6 expression was induced by ABA and drought treatments. The mutant cpk6 was insensitive to ABA treatment and low N, but was sensitive to drought only under high N conditions. CPK6 interacted with the NRT1.1 (CHL1) protein and phosphorylated the Thr447 residue, which then repressed the NO3- transporting activity of Arabidopsis under high N and drought stress. Taken together, our results show that CPK6 regulates Arabidopsis drought tolerance through changing the phosphorylation state of NRT1.1, and improve our knowledge of N uptake in plants during drought stress.

Characterization of the signalling pathways involved in the repression of root nitrate uptake by nitrate in Arabidopsis thaliana.

In Arabidopsis thaliana, root high-affinity nitrate (NO3-) uptake depends mainly on NRT2.1, 2.4, and 2.5, which are repressed by high NO3- supply at the transcript level. For NRT2.1, this regulation is due to the action of (i) feedback down-regulation by N metabolites and (ii) repression by NO3- itself mediated by the transceptor NRT1.1(NPF6.3). However, for NRT2.4 and NRT2.5, the signalling pathway(s) remain unknown as do the molecular elements involved. Here we show that unlike NRT2.1, NRT2.4 and NRT2.5 are not induced in an NO3- reductase mutant but are up-regulated following replacement of NO3- by ammonium (NH4+) as the N source. Moreover, increasing the NO3- concentration in a mixed nutrient solution with constant NH4+ concentration results in a gradual repression of NRT2.4 and NRT2.5, which is suppressed in an nrt1.1 mutant. This indicates that NRT2.4 and NRT2.5 are subjected to repression by NRT1.1-mediated NO3- sensing, and not to feedback repression by reduced N metabolites. We further show that key regulators of NRT2 transporters, such as HHO1, HRS1, PP2C, LBD39, BT1, and BT2, are also regulated by NRT1.1-mediated NO3- sensing, and that several of them are involved in NO3- repression of NRT2.1, NRT2.4, and NRT2.5. Finally, we provide evidence that it is the phosphorylated form of NRT1.1 at the T101 residue, which is most active in triggering the NRT1.1-mediated NO3- regulation of all these genes. Altogether, these data led us to propose a regulatory model for high-affinity NO3- uptake in Arabidopsis, highlighting several NO3- transduction cascades downstream of the phosphorylated form of the NRT1.1 transceptor.

A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase (msrA) Expression for Oxidative Stress Adaptation.

Reactive oxygen species (ROS) can cause destructive damage to biological macromolecules and protein dysfunction in bacteria. Methionine sulfoxide reductase (Msr) with redox-active Cys and/or seleno-cysteine (Sec) residues can restore physiological functions of the proteome, which is essential for oxidative stress tolerance of the extremophile Deinococcus radiodurans. However, the underlying mechanism regulating MsrA enzyme activity in D. radiodurans under oxidative stress has remained elusive. Here, we identified the function of MsrA in response to oxidative stress. msrA expression in D. radiodurans was significantly upregulated under oxidative stress. The msrA mutant showed a deficiency in antioxidative capacity and an increased level of dabsyl-Met-S-SO, indicating increased sensitivity to oxidative stress. Moreover, msrA mRNA was posttranscriptionally regulated by a small RNA, DsrO. Analysis of the molecular interaction between DsrO and msrA mRNA demonstrated that DsrO increased the half-life of msrA mRNA and then upregulated MsrA enzyme activity under oxidative stress compared to the wild type. msrA expression was also transcriptionally regulated by the DNA-repairing regulator DrRRA, providing a connection for further analysis of protein restoration during DNA repair. Overall, our results provide direct evidence that DsrO and DrRRA regulate msrA expression at two levels to stabilize msrA mRNA and increase MsrA protein levels, revealing the protective roles of DsrO signaling in D. radiodurans against oxidative stress. IMPORTANCE The repair of oxidized proteins is an indispensable function allowing the extremophile D. radiodurans to grow in adverse environments. Msr proteins and various oxidoreductases can reduce oxidized Cys and Met amino acid residues of damaged proteins to recover protein function. Consequently, it is important to investigate the molecular mechanism maintaining the high reducing activity of MsrA protein in D. radiodurans during stresses. Here, we showed the protective roles of an sRNA, DsrO, in D. radiodurans against oxidative stress. DsrO interacts with msrA mRNA to improve msrA mRNA stability, and this increases the amount of MsrA protein. In addition, we also showed that DrRRA transcriptionally regulated msrA gene expression. Due to the importance of DrRRA in regulating DNA repair, this study provides a clue for further analysis of MsrA activity during DNA repair. This study indicates that protecting proteins from oxidation is an effective strategy for extremophiles to adapt to stress conditions.

CALMODULIN-LIKE-38 and PEP1 RECEPTOR 2 integrate nitrate and brassinosteroid signals to regulate root growth.

Plants exhibit remarkable developmental plasticity, enabling them to adapt to adverse environmental conditions such as low nitrogen (N) in the soil. Brassinosteroids (BRs) promote root foraging for nutrients under mild N deficiency, but the crosstalk between the BR- and N-signaling pathways in the regulation of root growth remains largely unknown. Here, we show that CALMODULIN-LIKE-38 (CML38), a calmodulin-like protein, specifically interacts with the PEP1 RECEPTOR 2 (PEPR2), and negatively regulates root elongation in Arabidopsis (Arabidopsis thaliana) in response to low nitrate (LN). CML38 and PEPR2 are transcriptionally induced by treatments of exogenous nitrate and BR. Compared with Col-0, the single mutants cml38 and pepr2 and the double mutant cml38 pepr2 displayed enhanced primary root growth and produced more lateral roots under LN. This is consistent with their higher nitrate absorption abilities, and their stronger expression of nitrate assimilation genes. Furthermore, CML38 and PEPR2 regulate common downstream genes related to BR signaling, and they have positive roles in BR signaling. Low N facilitated BR signal transmission in Col-0 and CML38- or PEPR2-overexpressing plants, but not in the cml38 and pepr2 mutants. Taken together, our results illustrate a mechanism by which CML38 interacts with PEPR2 to integrate LN and BR signals for coordinating root development to prevent quick depletion of N resources in Arabidopsis.

Arabidopsis NRT1.2 interacts with the PHOSPHOLIPASE Dα1 (PLDα1) to positively regulate seed germination and seedling development in response to ABA treatment.

NRT1.2 has been characterized as a low-affinity nitrate transporter and an abscisic acid (ABA) transporter in Arabidopsis. In this study, we demonstrate that NRT1.2 positively regulated the ABA response during germination and seedling development. The transgenic Arabidopsis NRT1.2-over-expressionors showed increased sensitivity to ABA during these processes. qRT-PCR assays indicated that NRT1.2 over-production in 7-days-old seedlings up-regulated the expression of ABA-responsive genes: ABI1, ABI2, ABI3, ABI4, ABI5, RAB18, RD29A, and RD29B and PHOSPHOLIPASE Dα1 (PLDα1). The expression of these genes was suppressed in the nrt1.2 mutant in comparison with the wild type following ABA treatment. Importantly, bimolecular fluorescence complementation assays indicated that NRT1.2 interacts with PLDα1 at the plasma membrane. Their interaction was further confirmed by using yeast two hybrid (Y2H) experiments with the mating-based split ubiquitin system (MbSUS). Moreover, genetic assays indicated that PLDα1 acts epistatically on NRT1.2 to affect ABA signaling. Taken together, our results provide detailed mechanisms of NRT1.2 in ABA-mediated seed germination and seedling development.

The peptidyl-prolyl isomerases FKBP15-1 and FKBP15-2 negatively affect lateral root development by repressing the vacuolar invertase VIN2 in Arabidopsis.

MAIN CONCLUSION: The peptidyl-prolyl isomerases FKBP15-1 and FKBP15-2 negatively modulate lateral root development by repressing vacuolar invertase VIN2 activity. Lateral root (LR) architecture greatly affects the efficiency of nutrient absorption and the anchorage of plants. Although the internal phytohormone regulatory mechanisms that control LR development are well known, how external nutrients influence lateral root development remains elusive. Here, we characterized the function of two FK506-binding proteins, namely, FKBP15-1 and FKBP15-2, in Arabidopsis. FKBP15-1/15-2 genes were expressed prominently in the vascular bundles of the root basal meristem region, and the FKBP15-1/15-2 proteins were localized to the endoplasmic reticulum of the cells. Using IP-MS, Co-IP, and BiFC assays, we demonstrated that FKBP15-1 and FKBP15-2 interacted with vacuolar invertase 2 (VIN2). Compared to Col-0 and the single mutants, the fkbp15-1fkbp15-2 double mutant had more LRs, and presented higher sucrose catalytic activity. Moreover, genetic analysis showed genetic epistasis of VIN2 over FKBP15-1/FKBP15-2 in controlling LR development. Our results indicate that FKBP15-1 and FKBP15-2 participate in the control of LR number by inhibiting the catalytic activity of VIN2. Owing to the conserved peptidylprolyl cis-trans isomerase activity of FKBP family proteins, our results provide a clue for further analysis of the interplay between lateral root development and protein modification by FKBPs.

Cotton fiber elongation requires the transcription factor GhMYB212 to regulate sucrose transportation into expanding fibers.

Cotton is white gold across the globe and composed of fiber cells derived from the outer integument of cotton ovules. Fiber elongation uses sucrose as a direct carbon source. The molecular mechanism transcriptionally controlling sucrose transport from ovules into the elongating fibers remains elusive. In this study the involvement of GhMYB212 in the regulation of sucrose transportion into expanding fibers was investigated. GhMYB212 RNAi plants (GhMYB212i) accumulated less sucrose and glucose in developing fibers, and had shorter fibers and a lower lint index. RNA-seq and protein-DNA binding assays revealed that GhMYB212 was closely linked to the pathways of sucrose and starch transportation and metabolism, directly controling the expression of a sucrose transporter gene GhSWEET12. GhSWEET12 RNAi plants (GhSWEET12i) possessed similar fiber phenotypes to those of GhMYB212i. Exogenous sucrose supplementation in ovule cultures did not rescue the shorter fiber phenotype of GhMYB212i and GhSWEET12i. This finding supported the idea that the attenuated rate of sucrose transport from the outer seed coat into the fibers is responsible for the retardation of fiber elongation. Current investigations support the idea that GhMYB212 functions as the main regulator of fiber elongation by controlling the expression of GhSWEET12, and therefore it is important to study cell expansion and sugar transportation during seed development.

IBR5 Regulates Leaf Serrations Development via Modulation of the Expression of PIN1.

Biodiversity in plant shape is mainly attributable to the diversity of leaf shape, which is largely determined by the transient morphogenetic activity of the leaf margin that creates leaf serrations. However, the precise mechanism underlying the establishment of this morphogenetic capacity remains poorly understood. We report here that INDOLE-3-BUTYRIC ACID RESPONSE 5 (IBR5), a dual-specificity phosphatase, is a key component of leaf-serration regulatory machinery. Loss-of-function mutants of IBR5 exhibited pronounced serrations due to increased cell area. IBR5 was localized in the nucleus of leaf epidermis and petiole cells. Introducing a C129S mutation within the highly conserved VxVHCx2GxSRSx5AYLM motif of IBR5 rendered it unable to rescue the leaf-serration defects of the ibr5-3 mutant. In addition, auxin reporters revealed that the distribution of auxin maxima was expanded ectopically in ibr5-3. Furthermore, we found that the distribution of PIN1 on the plasma membrane of the epidermal and cells around the leaf vein was compromised in ibr5-3. We concluded that IBR5 is essential for the establishment of PIN-FORMED 1 (PIN1)-directed auxin maxima at the tips of leaf serration, which is vital for the elaborated regulation during its formation.

A computational interactome for prioritizing genes associated with complex agronomic traits in rice (Oryza sativa).

Rice (Oryza sativa) is one of the most important staple foods for more than half of the global population. Many rice traits are quantitative, complex and controlled by multiple interacting genes. Thus, a full understanding of genetic relationships will be critical to systematically identify genes controlling agronomic traits. We developed a genome-wide rice protein-protein interaction network (RicePPINet, http://netbio.sjtu.edu.cn/riceppinet) using machine learning with structural relationship and functional information. RicePPINet contained 708 819 predicted interactions for 16 895 non-transposable element related proteins. The power of the network for discovering novel protein interactions was demonstrated through comparison with other publicly available protein-protein interaction (PPI) prediction methods, and by experimentally determined PPI data sets. Furthermore, global analysis of domain-mediated interactions revealed RicePPINet accurately reflects PPIs at the domain level. Our studies showed the efficiency of the RicePPINet-based method in prioritizing candidate genes involved in complex agronomic traits, such as disease resistance and drought tolerance, was approximately 2-11 times better than random prediction. RicePPINet provides an expanded landscape of computational interactome for the genetic dissection of agronomically important traits in rice.

Genome-Wide Inference of Protein-Protein Interaction Networks Identifies Crosstalk in Abscisic Acid Signaling.

Protein-protein interactions (PPIs) are essential to almost all cellular processes. To better understand the relationships of proteins in Arabidopsis (Arabidopsis thaliana), we have developed a genome-wide protein interaction network (AraPPINet) that is inferred from both three-dimensional structures and functional evidence and that encompasses 316,747 high-confidence interactions among 12,574 proteins. AraPPINet exhibited high predictive power for discovering protein interactions at a 50% true positive rate and for discriminating positive interactions from similar protein pairs at a 70% true positive rate. Experimental evaluation of a set of predicted PPIs demonstrated the ability of AraPPINet to identify novel protein interactions involved in a specific process at an approximately 100-fold greater accuracy than random protein-protein pairs in a test case of abscisic acid (ABA) signaling. Genetic analysis of an experimentally validated, predicted interaction between ARR1 and PYL1 uncovered cross talk between ABA and cytokinin signaling in the control of root growth. Therefore, we demonstrate the power of AraPPINet (http://netbio.sjtu.edu.cn/arappinet/) as a resource for discovering gene function in converging signaling pathways and complex traits in plants.

Genome-wide cloning, identification, classification and functional analysis of cotton heat shock transcription factors in cotton (Gossypium hirsutum).

BACKGROUND: Heat shock transcriptional factors (Hsfs) play important roles in the processes of biotic and abiotic stresses as well as in plant development. Cotton (Gossypium hirsutum, 2n=4x=(AD)2=52) is an important crop for natural fiber production. Due to continuous high temperature and intermittent drought, heat stress is becoming a handicap to improve cotton yield and lint quality. Recently, the related wild diploid species Gossypium raimondii genome (2n=2x=(D5)2=26) has been fully sequenced. In order to analyze the functions of different Hsfs at the genome-wide level, detailed characterization and analysis of the Hsf gene family in G. hirsutum is indispensable. RESULTS: EST assembly and genome-wide analyses were applied to clone and identify heat shock transcription factor (Hsf) genes in Upland cotton (GhHsf). Forty GhHsf genes were cloned, identified and classified into three main classes (A, B and C) according to the characteristics of their domains. Analysis of gene duplications showed that GhHsfs have occurred more frequently than reported in plant genomes such as Arabidopsis and Populus. Quantitative real-time PCR (qRT-PCR) showed that all GhHsf transcripts are expressed in most cotton plant tissues including roots, stems, leaves and developing fibers, and abundantly in developing ovules. Three expression patterns were confirmed in GhHsfs when cotton plants were exposed to high temperature for 1 h. GhHsf39 exhibited the most immediate response to heat shock. Comparative analysis of Hsfs expression differences between the wild-type and fiberless mutant suggested that Hsfs are involved in fiber development. CONCLUSIONS: Comparative genome analysis showed that Upland cotton D-subgenome contains 40 Hsf members, and that the whole genome of Upland cotton contains more than 80 Hsf genes due to genome duplication. The expression patterns in different tissues in response to heat shock showed that GhHsfs are important for heat stress as well as fiber development. These results provide an improved understanding of the roles of the Hsf gene family during stress responses and fiber development.

Cotton microtubule-associated protein GhMAP20L5 mediates fiber elongation through the interaction with the tubulin GhTUB13.

Targeting proteins for Xklp2 (TPX2s) comprise a class of MAPs that are essential for plant growth and development by regulating the dynamic changes of microtubules (MTs) and proper formation of cytoskeleton. However, the function of TPX2 proteins in cotton fiber development remains poorly understood. Here, we identified the function of a fiber elongation-specific TPX2 protein, GhMAP20L5, in cotton. Suppressed GhMAP20L5 gene expression in cotton (GhMAP20L5i) significantly reduced fiber elongation rate, fiber length and lint percentage. GhMAP20L5i fibers had thinner and looser secondary cell walls (SCW), and incompact helix twists. GhMAP20L5 specifically interacted with the tubulin GhTUB13 on the cytoskeleton. Gene coexpression analysis showed that GhMAP20L5 involved in multiple pathways related to cytoskeleton establishment and fiber cell wall formation and affected cellulase genes expressions. In summary, our results revealed that GhMAP20L5 is important for fiber development by regulating cytoskeleton establishment and the cellulose deposition in cotton.

Designing artificial synthetic promoters for accurate, smart, and versatile gene expression in plants.

With the development of high-throughput biology techniques and artificial intelligence, it has become increasingly feasible to design and construct artificial biological parts, modules, circuits, and even whole systems. To overcome the limitations of native promoters in controlling gene expression, artificial promoter design aims to synthesize short, inducible, and conditionally controlled promoters to coordinate the expression of multiple genes in diverse plant metabolic and signaling pathways. Synthetic promoters are versatile and can drive gene expression accurately with smart responses; they show potential for enhancing desirable traits in crops, thereby improving crop yield, nutritional quality, and food security. This review first illustrates the importance of synthetic promoters, then introduces promoter architecture and thoroughly summarizes advances in synthetic promoter construction. Restrictions to the development of synthetic promoters and future applications of such promoters in synthetic plant biology and crop improvement are also discussed.

Genome-wide identification of nitrate transporter genes from Spirodela polyrhiza and characterization of SpNRT1.1 function in plant development.

Nitrate transporter (NRT) genes that participate in nitrate transport and distribution are indispensable for plant growth, development, and stress tolerance. Spirodela polyrhiza has the smallest genome among monocotyledon plants, and it has strong nitrate absorbance and phytoremediation abilities. However, the evolutionary history, expression patterns, and functions of the NRT gene family in S. polyrhiza are not well understood. Here, we identified 29 NRT members in the S. polyrhiza genome. Gene structure and phylogeny analyses showed that S. polyrhiza nitrate transporter (SpNRTs) genes were divided into eight clades without gene expansion compared with that in Arabidopsis. Transcriptomic analysis showed that SpNRT genes have spatiotemporal expression patterns and respond to abiotic stress. Functional analysis revealed that in S. polyrhiza, SpNRT1.1 expression was strongly induced by treatment with nitrate and ammonium. Overexpression of SpNRT1.1 significantly repressed primary root length, and the number and total length of lateral roots. This was more pronounced in high ammonium concentration medium. Overexpressed SpNRT1.1 in Arabidopsis significantly improved biomass and delayed flowering time, indicating that the nitrate transport ability of SpNRT1.1 differs from AtNRT1.1. In conclusion, our results provide valuable information about the evolution of the NRT family in higher plants and the function of SpNRT1.1.

Molecular cloning and expression analysis of a novel SANT/MYB gene from Gossypium barbadense.

MYB family transcription factors are implicated in multiple developmental processes. Herein, a new full-length cDNA encoding a SANT/MYB transcription factor (designated as GbRL2) was cloned and characterized from cotton (Gossypium barbadense L.) for the first time. The full-length cDNA of GbRL2 was 573 bp with a 240 bp open reading frame (ORF) encoding a deduced protein of 80 amino acid polypeptide with a calculated molecular mass of 8.96 kDa and an isoelectric point of 8.96. Sequence alignment revealed that GbRL2 had high homology with other single SANT/MYB domain containing genes, including the RADIALIS genes in Antirrhinum majus and Bournea leiophylla. Semi-quantitative reverse transcript polymerase chain reaction (RT-PCR) revealed that at seedling stage, GbRL2 was strongly expressed in leaves but merely in stems. In opening flowers, the expression of GbRL2 was moderate in the petals but could not be detected in stamens. In ovules, the expression of GbRL2 could not be detected at -3 days post-anthesis (DPA) but increased during early elongation stage (0 DPA, +3 DPA, +5 DPA and +8 DPA). The transcripts of GbRL2 could also be detected at +8 DPA elongating fibers. We also examined the expression of RL2 gene in Gossypium hirstum cultivar Xu-142 and its fuzzless-lintless-seed mutant fl plants. The GhRL2 gene was ectopically expressed at -3 DPA in the fl mutant while the expression of GhRL2 in WT could not be detected. The expression of GhRL2 decreased early (+5 DPA) while that of WT was still strong. Our results suggest that GbRL2 may participate in development of various organs and may be a target for genetic improvement of cotton fiber.

An L1 box binding protein, GbML1, interacts with GbMYB25 to control cotton fibre development.

Transcription factors play key roles in plant development through their interaction with cis-elements and/or other transcription factors. A HD-Zip IV family transcription factor, Gossypium barbadense Meristem Layer 1 (GbML1) has been identified and characterized here. GbML1 specifically bound to the L1 box and the promoters of GbML1 and GbRDL1. GbML1 physically interacted with a key regulator of cotton fibre development, GbMYB25. Truncated and point mutation assays indicated the START-SAD domain was required for the binding to the C terminal domain (CTD) of GbMYB25. GbML1 overexpression in Arabidopsis increased the number of trichomes on stems and leaves and increased the accumulation of anthocyanin in leaves. Taken together, the L1 box binding protein, GbML1 was identified as the first partner for GbMYB25 and the role of START domain was discovered to be a protein binding domain in plants. Our findings will help the improvement of cotton fibre production and the understanding of the key role of HD-Zip family and MYB family in plants.

Functional analysis of GbAGL1, a D-lineage gene from cotton (Gossypium barbadense).

Cotton fibres originate from the outer ovule integument and D-lineage genes are essential for ovule development and their roles can be described by the 'ABCDE' model of flower development. To investigate the role of D-lineage genes during ovule and fibre development, GbAGL1 (GenBank accession number: FJ198049) was isolated from G. barbadense by using the SMART RACE strategy. Sequence and phylogenetic analyses revealed that GbAGL1 was a member of the D-lineage gene family. Southern blot analysis showed that GbAGL1 belonged to a low-copy gene family. Semi-quantitative RT-PCR and RNA in situ hybridization analyses revealed that the GbAGL1 gene in G. barbadense was highly expressed in whole floral bud primordia and the floral organs including ovules and fibres, but the signals were barely observed in vegetative tissues. GbAGL1 expression increased gradually with the ovule developmental stages. Over-expression of GbAGL1 in Arabidopsis caused obvious homeotic alternations in the floral organs, such as early flowering, and an extruded stigma, which were the typical phenotypes of the D-lineage gene family. In addition, a complementation test revealed that GbAGL1 could rescue the phenotypes of the stk mutant. Our study indicated that GbAGL1 was a D-lineage gene that was involved in ovule development and might play key roles in fibres development.