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.

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus.

Ferulate-5-hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate-5-hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D-NMR profiling in KO-7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.

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.

Integrated genetic mapping and transcriptome analysis reveal the BnaA03.IAA7 protein regulates plant architecture and gibberellin signaling in Brassica napus L.

KEY MESSAGE: A novel mutation in the BnaA03.IAA7 protein reduces plant height and enhances gibberellin signaling in Brassica napus L. Rapeseed (Brassica napus) is an excellent and important source for vegetable oil production, but its production is severely affected by lodging. Lodging hinders mechanization and decreases yield, and an ideal solution is semidwarf breeding. Limited by germplasm resources, semidwarf breeding developed slowly in rapeseed. In the current study, a mutant called sdA03 was isolated from EMS-mutagenized lines of Zhongshuang 11 (ZS11). The inheritance analysis showed that phenotypes of sdA03 were controlled by a single semidominant gene. Genetic mapping, RNA-seq and candidate gene analysis identified BnaA03.IAA7 as a candidate gene, and a function test confirmed that the mutated BnaA03.iaa7 regulates plant architecture in a dose-dependent manner. Yeast two-hybrid and transient expression experiments illustrated the P87L substitution in the GWPPV/I degron motif of BnaA03.iaa7 impaired the interaction between BnaA03.IAA7 and TIR1 proteins, and BnaA03.iaa7 prevented ARF from activating the auxin signaling pathway.The gibberellin (GA) content was higher in sdA03 hypocotyls than in those of ZS11. Further expression analysis showed more active gibberellin signaling in hypocotyl and richer expression of GA synthetic genes in root and cotyledon of sdA03 seedlings. Finally, a marker was developed based on the SNP found in BnaA03.iaa7 and used in molecular breeding. The study enriched our understanding of the architectural regulation of rapeseed and provided germplasm resources for breeding.

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.

GhKWL1 Upregulates GhERF105 but Its Function Is Impaired by Binding with VdISC1, a Pathogenic Effector of Verticillium dahliae.

Verticillium wilt, caused by Verticillium dahliae, is a devastating disease for many important crops, including cotton. Kiwellins (KWLs), a group of cysteine-rich proteins synthesized in many plants, have been shown to be involved in response to various phytopathogens. To evaluate genes for their function in resistance to Verticillium wilt, we investigated KWL homologs in cotton. Thirty-five KWL genes (GhKWLs) were identified from the genome of upland cotton (Gossypium hirsutum). Among them, GhKWL1 was shown to be localized in nucleus and cytosol, and its gene expression is induced by the infection of V. dahliae. We revealed that GhKWL1 was a positive regulator of GhERF105. Silencing of GhKWL1 resulted in a decrease, whereas overexpression led to an increase in resistance of transgenic plants to Verticillium wilt. Interestingly, through binding to GhKWL1, the pathogenic effector protein VdISC1 produced by V. dahliae could impair the defense response mediated by GhKWL1. Therefore, our study suggests there is a GhKWL1-mediated defense response in cotton, which can be hijacked by V. dahliae through the interaction of VdISC1 with GhKWL1.

Cytokinin inhibits cotton fiber initiation by disrupting PIN3a-mediated asymmetric accumulation of auxin in the ovule epidermis.

Auxin-dependent cell expansion is crucial for initiation of fiber cells in cotton (Gossypium hirsutum), which ultimately determines fiber yield and quality. However, the regulation of this process is far from being well understood. In this study, we demonstrate an antagonistic effect between cytokinin (CK) and auxin on cotton fiber initiation. In vitro and in planta experiments indicate that enhanced CK levels can reduce auxin accumulation in the ovule integument, which may account for the defects in the fiberless mutant xu142fl. In turn, supplementation with auxin can recover fiber growth of CK-treated ovules and mutant ovules. We further found that GhPIN3a is a key auxin transporter for fiber-cell initiation and is polarly localized to the plasma membranes of non-fiber cells, but not to those of fiber cells. This polar localization allows auxin to be transported within the ovule integument while specifically accumulating in fiber cells. We show that CKs antagonize the promotive effect of auxin on fiber cell initiation by undermining asymmetric accumulation of auxin in the ovule epidermis through down-regulation of GhPIN3a and disturbance of the polar localization of the protein.

The phosphatidylinositol synthase gene (GhPIS) contributes to longer, stronger, and finer fibers in cotton.

Cotton fibers are the most important natural raw material used in textile industries world-wide. Fiber length, strength, and fineness are the three major traits which determine the quality and economic value of cotton. It is known that exogenous application of phosphatidylinositols (PtdIns), important structural phospholipids, can promote cotton fiber elongation. Here, we sought to increase the in planta production of PtdIns to improve fiber traits. Transgenic cotton plants were generated in which the expression of a cotton phosphatidylinositol synthase gene (i.e., GhPIS) was controlled by the fiber-specific SCFP promoter element, resulting in the specific up-regulation of GhPIS during cotton fiber development. We demonstrate that PtdIns content was significantly enhanced in transgenic cotton fibers and the elevated level of PtdIns stimulated the expression of genes involved in PtdIns phosphorylation as well as promoting lignin/lignin-like phenolic biosynthesis. Fiber length, strength and fineness were also improved in the transgenic plants as compared to the wild-type cotton, with no loss in overall fiber yield. Our data indicate that fiber-specific up-regulation of PtdIns synthesis is a promising strategy for cotton fiber quality improvement.

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.

Cotton sphingosine kinase GhLCBK1 participates in fiber cell elongation by affecting sphingosine-1-phophate and auxin synthesis.

Sphingolipids serve as essential components of biomembrane and possess significant bioactive properties. Sphingosine-1-phophate (S1P) plays a key role in plant resistance to stress, but its specific impact on plant growth and development remains to be fully elucidated. Cotton fiber cells are an ideal material for investigating the growth and maturation of plant cells. In this study, we examined the content and composition of sphingosine (Sph) and S1P throughout the progression of fiber cell development. The content of S1P elevated gradually during fiber elongation but declined during the transition stage. Exogenous application of S1P promoted fiber elongation while using of FTY720 (an antagonist of S1P), and DMS (an inhibitor of LCBK) hindered fiber elongation. Cotton Long Chain Base Kinase 1 (GhLCBK1) was notably expressed during the fiber elongation stage, containing all conserved domains of LCBK protein and localized in the endoplasmic reticulum. Overexpression GhLCBK1 increased the S1P content and promoted fiber elongation while retarded secondary cell wall (SCW) deposition. Conversely, downregulation of GhLCBK1 reduced the S1P levels, and suppressed fiber elongation, and accelerated SCW deposition. Transcriptome analysis revealed that upregulating GhLCBK1 or applying S1P induced the expression of GhEXPANSIN and auxin related genes. Furthermore, the levels of IAA were elevated and reduced in the fibers when up-regulating or down-regulating GhLCBK1, respectively. Our investigation demonstrated that GhLCBK1 and its product S1P facilitated the elongation of fiber cells by affecting auxin biosynthesis. This study contributes novel insights into the intricate regulatory pathways involved in fiber cell elongation, identifying GhLCBK1 as a potential target gene and laying the groundwork for enhancing fiber quality via genetic manipulation.

Single-nucleus sequencing deciphers developmental trajectories in rice pistils.

Angiosperms possess a life cycle with an alternation of sporophyte and gametophyte generations, which happens in plant organs like pistils. Rice pistils contain ovules and receive pollen for successful fertilization to produce grains. The cellular expression profile in rice pistils is largely unknown. Here, we show a cell census of rice pistils before fertilization through the use of droplet-based single-nucleus RNA sequencing. The ab initio marker identification validated by in situ hybridization assists with cell-type annotation, revealing cell heterogeneity between ovule- and carpel-originated cells. A comparison of 1N (gametophyte) and 2N (sporophyte) nuclei identifies the developmental path of germ cells in ovules with typical resetting of pluripotency before the sporophyte-gametophyte transition, while trajectory analysis of carpel-originated cells suggests previously neglected features of epidermis specification and style function. These findings gain a systems-level view of cellular differentiation and development of rice pistils before flowering and lay a foundation for understanding female reproductive development in plants.

Comparative genomic analyses reveal the genetic basis of the yellow-seed trait in Brassica napus.

Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in-depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47aZY821, but not the yellow-seed allele BnA09MYB47aGH06, promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops.

A knockout mutation in the lignin biosynthesis gene CCR1 explains a major QTL for acid detergent lignin content in Brassica napus seeds.

Seed coat phenolic compounds represent important antinutritive fibre components that cause a considerable reduction in value of seed meals from oilseed rape (Brassica napus). The nutritionally most important fibre compound is acid detergent lignin (ADL), to which a significant contribution is made by phenylpropanoid-derived lignin precursors. In this study, we used bulked-segregant analysis in a population of recombinant inbred lines (RILs) from a cross of the Chinese oilseed rape lines GH06 (yellow seed, low ADL) and P174 (black seed, high ADL) to identify markers with tight linkage to a major quantitative trait locus (QTL) for seed ADL content. Fine mapping of the QTL was performed in a backcross population comprising 872 BC(1)F(2) plants from a cross of an F(7) RIL from the above-mentioned population, which was heterozygous for this major QTL and P174. A 3:1 phenotypic segregation for seed ADL content indicated that a single, dominant, major locus causes a substantial reduction in ADL. This locus was successively narrowed to 0.75 cM using in silico markers derived from a homologous Brassica rapa sequence contig spanning the QTL. Subsequently, we located a B. rapa orthologue of the key lignin biosynthesis gene CINNAMOYL CO-A REDUCTASE 1 (CCR1) only 600 kbp (0.75 cM) upstream of the nearest linked marker. Sequencing of PCR amplicons, covering the full-length coding sequences of Bna.CCR1 homologues, revealed a locus in P174 whose sequence corresponds to the Brassica oleracea wild-type allele from chromosome C8. In GH06, however, this allele is replaced by a homologue derived from chromosome A9 that contains a loss-of-function frameshift mutation in exon 1. Genetic and physical map data infer that this loss-of-function allele has replaced a functional Bna.CCR1 locus on chromosome C8 in GH06 by homoeologous non-reciprocal translocation.

Loss of function of VdDrs2, a P4-ATPase, impairs the toxin secretion and microsclerotia formation, and decreases the pathogenicity of Verticillium dahliae.

Four P4-ATPase flippase genes, VdDrs2, VdNeo1, VdP4-4, and VdDnf1 were identified in Verticillium dahliae, one of the most devastating phytopathogenic fungi in the world. Knock out of VdDrs2, VdNeo1, and VdP4-4, or knock down of VdDnf1 significantly decreased the pathogenicity of the mutants in cotton. Among the mutants, the greatest decrease in pathogenicity was observed in ΔVdDrs2. VdDrs2 was localized to plasma membrane, vacuoles, and trans-Golgi network (TGN). In vivo observation showed that the infection of the cotton by ΔVdDrs2 was significantly delayed. The amount of two known Verticillium toxins, sulfacetamide, and fumonisin B1 in the fermentation broth produced by the ΔVdDrs2 strain was significantly reduced, and the toxicity of the crude Verticillium wilt toxins to cotton cells was attenuated. In addition, the defect of VdDrs2 impaired the synthesis of melanin and the formation of microsclerotia, and decreased the sporulation of V. dahliae. Our data indicate a key role of P4 ATPases-associated vesicle transport in toxin secretion of disease fungi and support the importance of mycotoxins in the pathogenicity of V. dahliae.

Arabidopsis P4 ATPase-mediated cell detoxification confers resistance to Fusarium graminearum and Verticillium dahliae.

Many toxic secondary metabolites produced by phytopathogens can subvert host immunity, and some of them are recognized as pathogenicity factors. Fusarium head blight and Verticillium wilt are destructive plant diseases worldwide. Using toxins produced by the causal fungi Fusarium graminearum and Verticillium dahliae as screening agents, here we show that the Arabidopsis P4 ATPases AtALA1 and AtALA7 are responsible for cellular detoxification of mycotoxins. Through AtALA1-/AtALA7-mediated vesicle transport, toxins are sequestered in vacuoles for degradation. Overexpression of AtALA1 and AtALA7 significantly increases the resistance of transgenic plants to F. graminearum and V. dahliae, respectively. Notably, the concentration of deoxynivalenol, a mycotoxin harmful to the health of humans and animals, was decreased in transgenic Arabidopsis siliques and maize seeds. This vesicle-mediated cell detoxification process provides a strategy to increase plant resistance against different toxin-associated diseases and to reduce the mycotoxin contamination in food and feed.

Localization of QTLs for seed color using recombinant inbred lines of Brassica napus in different environments.

Yellow seed is one of the most important traits of Brassica napus L. Efficient selection of the yellow-seed trait is one of the most important objectives in oilseed rape breeding. Two recombinant inbred line (RIL) populations (RIL-1 and RIL-2) were analyzed for 2 years at 2 locations. Four hundred and twenty SSR, RAPD, and SRAP marker loci covering 1744 cM were mapped in 26 linkage groups of RIL-1, while 265 loci covering 1135 cM were mapped in 20 linkage groups of RIL-2. A total of 19 QTLs were detected in the 2 populations. A major QTL was detected adjacent to the same marker (EM11ME20/200) in both maps in both years. This major QTL could explain 53.71%, 39.34%, 42.42%, 30.18%, 24.86%, and 15.08% of phenotypic variation in 6 combinations (location x year x population). BLASTn analysis of the sequences of the markers flanking the major QTL revealed that the homologous region corresponding to this major QTL was anchored between genes At5g44440 and At5g49640 of Arabidopsis thaliana chromosome 5 (At C5). Based on comparative genomic analysis, the bifunctional gene TT10 is nearest to the homologue of EM11ME20/200 on At C5 and can be considered an important candidate gene for the major QTL identified here. Besides providing an effective strategy for marker-assisted selection of the yellow-seed trait in B. napus, our results also provide important clues for cloning of the candidate gene corresponding to this major QTL.