R2R3 MYB Transcription Factor GhMYB201 Promotes Cotton Fiber Elongation via Cell Wall Loosening and Very-Long-Chain Fatty Acid Synthesis.

Cotton fiber is the leading natural textile material, and fiber elongation plays an essential role in the formation of cotton yield and quality. Although a number of components in the molecular network controlling cotton fiber elongation have been reported, a lot of players still need to be functionally dissected to understand the regulatory mechanism of fiber elongation comprehensively. In the present study, an R2R3-MYB transcription factor gene, GhMYB201, was characterized and functionally verified via CRISPR/Cas9-mediated gene editing. GhMYB201 was homologous to Arabidopsis AtMYB60, and both coding genes (GhMYB201At and GhMYB201Dt) were preferentially expressed in elongating cotton fibers. Knocking-out of GhMYB201 significantly reduced the rate and duration of fiber elongation, resulting in shorter and coarser mature fibers. It was found that GhMYB201 could bind and activate the transcription of cell wall loosening genes (GhRDLs) and also ß-ketoacyl-CoA synthase genes (GhKCSs) to enhance very-long-chain fatty acid (VLCFA) levels in elongating fibers. Taken together, our data demonstrated that the transcription factor GhMYB201s plays an essential role in promoting fiber elongation via activating genes related to cell wall loosening and VLCFA biosynthesis.

GhROP6 GTPase modulates auxin accumulation in cotton fibers by regulating cell-specific GhPIN3a localization.

PIN-FORMED- (PIN) mediated polar auxin transport plays a predominant role in most auxin-triggered organogenesis in plants. Global control of PIN polarity at the plasma membrane contributes to the essential establishment of auxin maxima in most multicellular tissues. However, establishment of auxin maxima in single cells is poorly understood. Cotton fibers, derived from ovule epidermal cells by auxin-triggered cell protrusion, provide an ideal model to explore the underlying mechanism. Here, we report that cell-specific degradation of GhPIN3a, which guides the establishment of the auxin gradient in cotton ovule epidermal cells, is associated with the preferential expression of GhROP6 GTPase in fiber cells. In turn, GhROP6 reduces GhPIN3a abundance at the plasma membrane and facilitates intracellular proteolysis of GhPIN3a. Overexpression and activation of GhROP6 promote cell elongation, resulting in a substantial improvement in cotton fiber length.

Yellow Petal locus GaYP promotes flavonol biosynthesis and yellow coloration in petals of Asiatic cotton (Gossypium arboreum).

KEY MESSAGE: Yellow Petal locus GaYP is located on chromosome 11 and encodes a Sg6 R2R3-MYB transcription factor, which promotes flavonol biosynthesis and yellow coloration in Asiatic cotton petals. Petal color is pivotal to ornamental value and reproduction of plants. Yellow coloration in plant petals is mainly attributed to colorants including carotenoids, aurones and some flavonols. To date, the genetic regulatory mechanism of flavonol biosynthesis in petals is still to be elucidated. Here, we employed Asiatic cottons with or without deep yellow coloration in petals to address this question. Multi-omic and biochemical analysis revealed significantly up-regulated transcription of flavonol structural genes and increased levels of flavonols, especially gossypetin and 6-hydroxykaempferol, in yellow petals of Asiatic cotton. Furthermore, the Yellow Petal gene (GaYP) was mapped on chromosome 11 by using a recombinant inbred line population. It was found that GaYP encoded a transcriptional factor belonging to Sg6 R2R3-MYB proteins. GaYP could bind to the promoter of flavonol synthase gene (GaFLS) and activate the transcription of downstream genes. Knocking out of GaYP or GaFLS homologs in upland cotton largely eliminated flavonol accumulation and pale yellow coloration in petals. Our results indicated that flavonol synthesis, up-regulated by the R2R3-MYB transcription activator GaYP, was the causative factor for yellow coloration of Asiatic cotton petals. In addition, knocking out of GaYP homologs also led to decrease in anthocyanin accumulation and petal size in upland cotton, suggesting that GaYP and its homologs might modulate developmental or physiological processes beyond flavonol biosynthesis.

Genome resequencing-based high-density genetic map and QTL detection for yield and fiber quality traits in diploid Asiatic cotton (Gossypium arboreum).

Cotton is the most important fiber crop in the world. Asiatic cotton (Gossypium arboreum, genome A2) is a diploid cotton species producing spinnable fibers and important germplasm for cotton breeding and a significant model for fiber biology. However, the genetic map of Asiatic cotton has been lagging behind tetraploid cottons, as well as other stable crops. This study aimed to construct a high-density SNP genetic map and to map QTLs for important yield and fiber quality traits. Using a recombinant inbred line (RIL) population and genome resequencing technology, we constructed a high-density genetic map that covered 1980.17 cM with an average distance of 0.61 cM between adjacent markers. QTL analysis revealed a total of 297 QTLs for 13 yield and fiber quality traits in three environments, explaining 5.0-37.4% of the phenotypic variance, among which 75 were stably detected in two or three environments. Besides, 47 QTL clusters, comprising 131 QTLs for representative traits, were identified. Our works laid solid foundation for fine mapping and cloning of QTL for yield and fiber quality traits in Asiatic cotton.

Carpel-specific down-regulation of GhCKXs in cotton significantly enhances seed and fiber yield.

Cytokinin is considered to be an important driver of seed yield. To increase the yield of cotton while avoiding the negative consequences caused by constitutive overproduction of cytokinin, we down-regulated specifically the carpel genes for cytokinin oxidase/dehydrogenase (CKX), a key negative regulator of cytokinin levels, in transgenic cotton. The carpel-specific down-regulation of CKXs significantly enhanced cytokinin levels in the carpels. The elevated cytokinin promoted the expression of carpel- and ovule-development-associated genes, GhSTK2, GhAG1, and GhSHP, boosting ovule formation and thus producing more seeds in the ovary. Field experiments showed that the carpel-specific increase of cytokinin significantly increased both seed yield and fiber yield of cotton, without resulting in detrimental phenotypes. Our study details the regulatory mechanism of cytokinin signaling for seed development, and provides an effective and feasible strategy for yield improvement of seed crops.

Microtubules play a crucial role in regulating actin organization and cell initiation in cotton fibers.

KEY MESSAGE: Dynamic organization of actin and microtubule cytoskeletons directs a distinct expansion behavior of cotton fiber initiation from cell elongation. Cotton fibers are highly elongated single cells derived from the ovule epidermis. Although actin and microtubule (MT) cytoskeletons have been implicated in cell elongation and secondary wall deposition, their roles in fiber initiation is poorly understood. Here, we used fluorescent probes and pharmacological approaches to study the roles of these cytoskeletal components during cotton fiber initiation. Both cytoskeletons align along the growth axis in initiating fibers. The dorsal view of ovules shows that unlike the fine actin filaments (AFs) in nonfiber cells, the AFs in fiber cells are dense and bundled. MTs are randomized in fiber cells and well-ordered in nonfiber cells. The pharmacological experiments revealed that the depolymerization of AFs and MTs assisted fiber initiation. Both AF stabilization and depolymerization inhibited fiber elongation. In contrast, the proper depolymerization of MTs promoted cell elongation, although the MT-stabilizing drug consistently resulted in a negative effect. Notably, we found that the organization of AFs was correlated with MT dynamics. Stabilizing the MTs by taxol treatment promoted the formation of AF bundles (in fiber initials) and transversely aligned AFs (in elongating fibers), whereas depolymerizing the MTs by oryzalin treatment promoted the fragmentation of AFs. Collectively, our data indicates that MTs plays a crucial role in regulating AF organization and early development of cotton fibers.

Circ_0047921 acts as the sponge of miR-1287-5p to stimulate lung cancer progression by regulating proliferation, migration, invasion, and glycolysis of lung cancer cells.

BACKGROUND: Lung cancer is a common respiratory system disease caused by multiple factors. Circular RNAs (circRNAs) play vital roles in tumorigenesis, including lung cancer. This study aimed to clarify the role and underlying molecular mechanisms of circ_0047921 in lung cancer. METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR) was used to assess the expression levels of circ_0047921, La-related protein 1 (LARP1), and miR-1287-5p. Cell proliferation was analyzed by CCK-8 and EdU assays. Transwell assay was used to assess migration and invasion. Western blot assay was employed to quantify protein expression. Glycolysis ability of cell was determined by measuring glucose consumption and lactate production with matched kits. The relationship between miR-1287-5p and circ_0047921 or LARP1 was confirmed by dual-luciferase reporter assay. In addition, a xenograft model was established to clarify the functional role of circ_0047921 in vivo. RESULTS: Circ_0047921 was highly expressed in lung cancer tissues and cells. Circ_0047921 downregulation repressed proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) and glycolysis in lung cancer cells. Circ_0047921 targeted miR-1287-5p to deplete miR-1287-5p expression. The effects caused by circ_0047921 downregulation were reversed by miR-1287-5p inhibition. In addition, LARP1 was a target of miR-1287-5p, and circ_0047921 could directly interact with miR-1287-5p to increase the expression of LARP1. The effects caused by circ_0047921 downregulation were also reversed by LARP1 overexpression. Circ_0047921 silencing impeded the growth of tumor in vivo. CONCLUSION: Circ_0047921 was overexpressed in lung cancer, and circ_0047921 targeted miR-1287-5p to modulate LARP1 expression, thereby facilitating the development of lung cancer. TRIAL REGISTRATION: The present study was approved by the ethical review committee of The First People's Hospital of Chenzhou, Southern Medical University with reference no. 20210106.

Up-regulation of GhPAP1A results in moderate anthocyanin accumulation and pigmentation in sub-red cotton.

Anthocyanins are a group of important secondary metabolites, functioning as colorant in plant organs as well as protective agents against several stresses. Sub-red plant (Rs) cottons, accumulating moderate level of anthocyanins in shoots, had increased photosynthesis efficiency compared to green- (GL) and red-plant (R1) cottons. The present work aimed to clarify the molecular base of anthocyanin regulation in Rs cotton. It was found that GhPAP1A was significantly up-regulated in Rs plants compared to GL cottons, but its expression level is lower than that of GhPAP1D in R1 plants. Virus induced gene silencing of GhPAP1s inhibited the red pigmentation in Rs plants. Comparative cloning revealed a 50-bp tandem repeat in the promoter of GhPAP1A in Rs cotton, which showed stronger activity to drive the expression of downstream genes in petals. Considered that the coding sequence of GhPAP1As from Rs and GL cottons had similar functions to promote anthocyanin biosynthesis in transgenic tobaccos, we attributed moderate anthocyanin accumulation in Rs cotton to increased transcription of GhPAP1A, resulted from varied promoter structure. Our works suggested GhPAP1s as useful tool to manipulate anthocyanin level and several breeding targets, including herbivore- and pathogen- resistance, high photosynthesis efficiency and colored fibers.

Over-expression of the red plant gene R1 enhances anthocyanin production and resistance to bollworm and spider mite in cotton.

Anthocyanins are a class of pigments ubiquitously distributed in plants and play roles in adoption to several stresses. The red plant gene (R1) promotes light-induced anthocyanin accumulation and red/purple pigmentation in cotton. Using 11 markers developed via genome resequencing, the R1 gene was located in an interval of approximately 136 kb containing three annotated genes. Among them, a PAP1 homolog, GhPAP1D (Gohir.D07G082100) displayed differential transcript level in the red- and green-plant leaves. GhPAP1D encoded a R2R3-MYB transcription factor and its over-expression resulted in increased anthocyanin accumulation in transgenic tobaccos and cottons. Dual luciferase assay indicated that GhPAP1D activated the promoters of several cotton anthocyanin structural genes in tobacco leaves. Importantly, we found that the GhPAP1D-overexpressing cotton leaves had increased resistance to both bollworm and spite mite. Our data demonstrated that GhPAP1D was the controlling gene of the red plant phenotype in cotton, and as the major anthocyanin regulator, this gene was potential to create transgenic cottons with resistance to a broad spectrum of herbivores.

Natural variation in a CENTRORADIALIS homolog contributed to cluster fruiting and early maturity in cotton.

BACKGROUND: Plant architecture and the vegetative-reproductive transition have major impacts on the agronomic success of crop plants, but genetic mechanisms underlying these traits in cotton (Gossypium spp.) have not been identified. RESULTS: We identify four natural mutations in GoCEN-Dt associated with cluster fruiting (cl) and early maturity. The situ hybridization shows that GhCEN is preferentially expressed in cotton shoot apical meristems (SAM) of the main stem and axillary buds. Constitutive GhCEN-Dt overexpression suppresses the transition of the cotton vegetative apex to a reproductive shoot. Silencing GoCEN leads to early flowering and determinate growth, and in tetraploids causes the main stem to terminate in a floral bud, a novel phenotype that exemplifies co-adaptation of polyploid subgenomes and suggests new research and/or crop improvement approaches. Natural cl variations are enriched in cottons adapted to high latitudes with short frost-free periods, indicating that mutants of GoCEN have been strongly selected for early maturity. CONCLUSION: We show that the cotton gene GoCEN-Dt, a homolog of Antirrhinum CENTRORADIALIS, is responsible for determinate growth habit and cluster fruiting. Insight into the genetic control of branch and flower differentiation offers new approaches to develop early maturing cultivars of cotton and other crops with plant architecture appropriate for mechanical harvesting.

A D-genome-originated Ty1/Copia-type retrotransposon family expanded significantly in tetraploid cottons.

Retrotransposons comprise of a major fraction of higher plant genomes, and their proliferation and elimination have profound effects on genome evolution and gene functions as well. Previously we found a D-genome-originated Ty1/Copia-type LTR (DOCL) retrotransposon in the chromosome A08 of upland cotton. To further characterize the DOCL retrotransposon family, a total of 342 DOCL retrotransposons were identified in the sequenced cotton genomes, including 73, 157, and 112 from Gossypium raimondii, G. hirsutum, and G. barbadense, respectively. According to phylogenetic analysis, the DOCL family was divided into nine groups (G1-G9), among which five groups (G1-G4 and G9, including 292 members) were proliferated after the formation of tetraploid cottons. It was found that the majority of DOCL retrotransposons (especially those in G2, G3 and G9) inserted in non-allelic loci in G. hirsutum and G. barbadense, suggesting that their proliferations were relatively independent in different tetraploid cottons. Furthermore, DOCL retrotransposons inserted in coding regions largely eliminated expression of the targeted genes in G. hirsutum or G. barbadense. Our data suggested that recent proliferation of retrotransposon families like DOCL was one of important evolutionary forces driving diversification and evolution of tetraploid cottons.

Up-regulation of GhTT2-3A in cotton fibres during secondary wall thickening results in brown fibres with improved quality.

Brown cotton fibres are the most widely used naturally coloured raw materials for the eco-friendly textile industry. Previous studies have indicated that brown fibre pigments belong to proanthocyanidins (PAs) or their derivatives, and fibre coloration is negatively associated with cotton productivity and fibre quality. To date, the molecular basis controlling the biosynthesis and accumulation of brown pigments in cotton fibres is largely unknown. In this study, based on expressional and transgenic analyses of cotton homologs of ArabidopsisPA regulator TRANSPARENT TESTA 2 (TT2) and fine-mapping of the cotton dark-brown fibre gene (Lc1), we show that a TT2 homolog, GhTT2-3A, controls PA biosynthesis and brown pigmentation in cotton fibres. We observed that GhTT2-3A activated GhbHLH130D, a homolog of ArabidopsisTT8, which in turn synergistically acted with GhTT2-3A to activate downstream PA structural genes and PA synthesis and accumulation in cotton fibres. Furthermore, the up-regulation of GhTT2-3A in fibres at the secondary wall-thickening stage resulted in brown mature fibres, and fibre quality and lint percentage were comparable to that of the white-fibre control. The findings of this study reveal the regulatory mechanism controlling brown pigmentation in cotton fibres and demonstrate a promising biotechnological strategy to break the negative linkage between coloration and fibre quality and/or productivity.

Auxin Regulates Cotton Fiber Initiation via GhPIN-Mediated Auxin Transport.

Cotton fibers are seed trichomes that make cotton unique compared with other plants. At anthesis, IAA, a major auxin in plants, accumulates in the fiber cell to promote cell initiation. However, many important aspects of this process are not clear. Here, auxin distribution patterns indicated by auxin-dependent DR5::GUS (ß-glucuronidase) expression in cotton ovules were studied during fiber cell differentiation and cell initiation [-2 to 2 DPA (days post-anthesis)]. The nucellus and fiber cell were two major sites where auxin accumulates. The accumulation in the nucellus started from -1 DPA, and that in fiber cells from 0 DPA. Immunolocalization analysis further suggests that the IAA accumulation in fiber initials began before flower opening. Furthermore, we demonstrate that accumulated IAA in fiber initials was mainly from efflux transport and not from in situ synthesis. Eleven auxin efflux carrier (GhPIN) genes were identified, and their expression during ovule and fiber development was investigated. Ovule-specific suppression of multiple GhPIN genes in transgenic cotton inhibited both fiber initiation and elongation. In 0 DPA ovules, GhPIN3a, unlike other GhPIN genes, showed additional localization of the transcript in the outer integument. Collectively, these results demonstrate the important role of GhPIN-mediated auxin transport in fiber-specific auxin accumulation for fiber initiation.

[5-aza-2'-deoxycytidine suppresses the growth of human lung adenocarcinoma cells in nude mouse xenograft models and its effect on methylation status and expression of TFPI-2 gene].

OBJECTIVE: To investigate the inhibitory effect of classic demethylating drug 5-aza-2'-deoxycytidine (5-Aza-CdR) on the growth of human lung adenocarcinoma cells in nude mouse xenograft models, and to observe its effect on methylation status and expression of TFPI-2 gene in the nude mouse xenograft tissues. METHODS: The nude mouse xenograft model was established by subcutaneous inoculation of human lung adenocarcinoma A549 cells. According to different doses of 5-Aza-CdR, the tumor-bearing nude mice were randomly divided into experimental groups (0.5 mg/kg group, 1 mg/kg group, 2 mg/kg group) and control group (0 mg/kg group). The tumor growth in the nude mice was observed. The methylation status and the expression of TFPI-2 gene mRNA and protein were detected by methylation specific polymerase chain reaction, real-time fluorescent quantitative polymerase chain reaction and Western blot assay. RESULTS: The nude mice were euthanized at 28 days after intraperitoneal injection of 5-Aza-CdR. The body weight of tumor-bearing nude mice was (27.12 ± 0.38) g in the 0 mg/kg group, (26.80 ± 0.18) g in the 0.5 mg/kg group, (26.67 ± 0.28) g in the 1 mg/kg group, and (26.50 ± 0.26) g in the 2 mg/kg group, showing no significant difference among them (P > 0.05). The volume of xenograft tumors in the 0 mg/kg group was (709.22 ± 2.87)mm³, (400.67 ± 2.68)mm³ in the 0.5 mg/kg group, (285.71 ± 2.91)mm³ in the 1 mg/kg group, and (230.44 ± 3.15)mm³ in the 2 mg/kg group, showing a significant difference (P < 0.05). There were complete methylation of TFPI-2 gene in the 0 mg/kg group, incomplete methylation in the 0.5 and 1 mg/kg groups, and unmethylation in the 2 mg/kg group. The relative mRNA level in the 0, 0.5, 1, 2 mg/kg groups were 1.00 ± 0.00, 1.67 ± 0.07, 3.40 ± 0.24, and 5.55 ± 0.61, respectively (P < 0.05). The relative expression level of TFPI-2 protein in the 0, 0.5, 1, 2 mg/kg groups was 0.18 ± 0.02, 0.36 ± 0.01, 0.64 ± 0.02, and 0.81 ± 0.20, respectively (P < 0.05). CONCLUSIONS: 5-Aza-CdR suppresses the tumor growth of human lung adenocarcinoma cells in nude mouse xenograft models, and induces expression of TFPI-2 gene in the xenograft tumor cells. The mechanism might be that 5-Aza-CdR induces re-expression of demethylated TFPI-2 gene by demethylation, and thus inhibits the growth and proliferation of human lung adenocarcinoma cells.

BpAFP, a Broussonetia papyrifera latex chitinase, exhibits a dual role in resisting to both Verticillium wilt disease and lepidopterous pests, Plutella xylostella and Prodenia litura.

Paper mulberry (Broussonetia papyrifera) is a fast-growing tree known for its tolerance to diverse biotic and abiotic stresses. To explore genes combating Verticillium wilt, a devasting and formidable disease damage to cotton and many economically significant crops, we purified an antifungal protein, named BpAFP, from the latex of paper mulberry. Based on peptide fingerprint, we cloned the full cDNA sequence of BpAFP and revealed that BpAFP belongs to Class I chitinases, sharing 74 % identity with B. papyrifera leaf chitinase, PMAPII. We further introduced BpAFP into Arabidopsis, tobacco, and cotton. Transgenic plants exhibited significant resistance to Verticillium wilt. Importantly, BpAFP also demonstrated insecticidal activity against herbivorous pests, Plutella xylostella, and Prodenia litura, when feeding the larvae with transgenic leaves. Our finding unveils a dual role of BpAFP in conferring resistance to both plant diseases and lepidopterous pests.

Synthetic dual hormone-responsive promoters enable engineering of plants with broad-spectrum resistance.

In plant immunity, the mutually antagonistic hormones salicylic acid (SA) and jasmonic acid (JA) are implicated in resistance to biotrophic and necrotrophic pathogens, respectively. Promoters that can respond to both SA and JA signals are urgently needed to engineer plants with enhanced resistance to a broad spectrum of pathogens. However, few natural pathogen-inducible promoters are available for this purpose. To address this problem, we have developed a strategy to synthesize dual SA- and JA-responsive promoters by combining SA- and JA-responsive cis elements based on the interaction between their cognate trans-acting factors. The resulting promoters respond rapidly and strongly to both SA and Methyl Jasmonate (MeJA), as well as different types of phytopathogens. When such a synthetic promoter was used to control expression of an antimicrobial peptide, transgenic plants displayed enhanced resistance to a diverse range of biotrophic, necrotrophic, and hemi-biotrophic pathogens. A dual-inducible promoter responsive to the antagonistic signals auxin and cytokinin was generated in a similar manner, confirming that our strategy can be used for the design of other biotically or abiotically inducible systems.

DELLA-NAC Interactions Mediate GA Signaling to Promote Secondary Cell Wall Formation in Cotton Stem.

Gibberellins (GAs) promote secondary cell wall (SCW) development in plants, but the underlying molecular mechanism is still to be elucidated. Here, we employed a new system, the first internode of cotton, and the virus-induced gene silencing method to address this problem. We found that knocking down major DELLA genes via VIGS phenocopied GA treatment and significantly enhanced SCW formation in the xylem and phloem of cotton stems. Cotton DELLA proteins were found to interact with a wide range of SCW-related NAC proteins, and virus-induced gene silencing of these NAC genes inhibited SCW development with downregulated biosynthesis and deposition of lignin. The findings indicated a framework for the GA regulation of SCW formation; that is, the interactions between DELLA and NAC proteins mediated GA signaling to regulate SCW formation in cotton stems.

Specific Upregulation of a Cotton Phytoene Synthase Gene Produces Golden Cottonseeds with Enhanced Provitamin A.

Provitamin A (PVA) bio-fortification of crops offers a sustainable strategy to prevent the prevalence of vitamin A deficiency (VAD), one of the world's major public health problems. The present work aimed to enhance PVA accumulation in cottonseed, the main by-product in the production of cotton fibers and the third largest source of edible plant oil in the world. On the basis of comprehensive identification of carotenoid synthase genes and their expression levels in various cotton tissues, we selected phytoene synthase as the target for manipulating carotenoid biosynthesis in the developing cottonseeds. After functional verification in transgenic tobacco, a cotton phytoene synthase gene (GhPSY2D) driven by a seed-specific promoter was transformed into cotton. The transgenic cottonseeds showed golden appearance and contained over 6-fold higher carotenoid contents in the extracted oil than the non-transgenic control. Thin layer chromatograph analysis indicated that the main PVA carotenoid ß-carotene was predominant in the transgenic cottonseeds, but undetectable in the wild-type control. By simultaneously providing economically valuable fibers and edible oils, the transgenic cottons bio-fortified with ß-carotene in seeds may be a new powerful tool against VAD in low-income regions.

Sequential amplification of flanking sequences by Y-shaped adaptor dependent extension using multiple templates.

Y-shaped adaptor dependent extension (YADE) method is a useful tool to amplify the flanking sequence of a known DNA sequence, but its efficiency is frequently limited by the restriction sites around the known sequence. In this paper, we demonstrated that using multiple templates derived from several restrictions and ligations could dramatically increase the efficiency of YADE method and render it suitable for sequential amplification of flanking sequences. With templates originating from 7 digestions, a 2,228-bp 5'-upstream sequence of a cotton small GTPase gene was obtained by two rounds of sequential YADE amplifications. The results demonstrated that the YADE method with multiple templates may be a useful tool for sequential PCR walking in complex genomes.

PIN-formed protein, a door to reveal the mechanism for auxin-triggered initiation of cotton fiber.

Cotton fibers are differentiated ovule epidermal cells that provide an ideal model to study cell differentiation and elongation. Establishment of auxin maximum in fiber cells is crucial for cotton-fiber protrusion from ovule surface. However, it is unclear where the auxin originates from and how the auxin accumulates in fiber cells. Our recent results indicate that the auxin is mainly imported from the outside of ovules, and transported to fiber cells through GhPIN (homolog of PIN-formed proteins in cotton) -mediated polar auxin transport, rather than in situ synthesis. Based on our finding in GhPINs, we discuss here briefly how auxin flow to fiber cells and auxin gradient in ovule epidermis is established mainly by GhPIN3a protein.