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.

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.

Auxin Directly Upregulates GhRAC13 Expression to Promote the Onset of Secondary Cell Wall Deposition in Cotton Fibers.

Cotton fibers are single cells that show a relatively independent developmental process of cell differentiation, elongation, and secondary wall deposition. Auxin promotes fiber cell protrusion from the surface of the ovule. However, the role of auxin at other stages of cotton fiber development remains largely unknown. To gain a deeper insight into this aspect, we measured indoleacetic acid (IAA) content in developing fibers. Results showed an increase in IAA content at the transition stage from elongation to secondary cell wall deposition. Subsequently, we investigated the differences between two transgenic cottons that show upregulated and downregulated fiber auxin levels, respectively. In planta analysis revealed that, in addition to promoting cell elongation, auxin regulated the time of initiation of reactive oxygen species (ROS) production and secondary wall deposition in cotton fibers. This was closely correlated with the upregulated expression of GhRAC13, which regulates ROS-triggered cellulose synthesis. We found multiple putative auxin-responsive elements existed within the promoter region of GhRAC13, and IAA could induce proGhRAC13 activity. The dual-luciferase reporter assay further proved the activation of proGhRAC13 by GhARF5, an auxin-signaling activator. Altogether, our results suggest a role of auxin in promoting the onset of secondary growth by directly upregulating GhRAC13 expression in cotton fibers.

Senescence-induced expression of ZmSUT1 in cotton delays leaf senescence while the seed coat-specific expression increases yield.

KEY MESSAGE: Sink-specific expression of a sucrose transporter protein gene from the C4 plant maize can promote carbohydrate accumulation in target tissues and increase both fiber and seed yield of cotton. Sucrose is the principal form of photosynthetic products transported from source tissue to sink tissue in higher plants. Enhancing the partition of carbohydrate to the target organ is a promising way to improve crop productivity. The C4 plant Zea mays exhibits a substantially higher rate of export of photosynthates than many C3 plants, and its sucrose transporter protein ZmSut1 displays important role in sucrose allocation. To investigate how use of ZmSUT1 gene to increase the fiber and seed yield of cotton, in this study, we expressed the gene in cotton under a senescence-inducible promoter PSAG12 and a seed coat-specific promoter BAN, respectively. We show that senescence-induced expression of ZmSUT1 results in an increase of sugar accumulation in leaves. Although the leaf senescence was postponed in PSAG12::ZmSUT1 cotton, the photosynthetic rate of the leaves was decreased. In contrast, seed coat-specific expression of the gene leads to an increase of sugar accumulation in fibers and bolls, and the leaf of transgenic BAN::ZmSUT1 cotton displayed higher photosynthetic capacity than the wild type. Importantly, both fiber and seed yield of transgenic BAN::ZmSUT1 cotton are significantly enhanced. Our data indicate the potential of enhancing yield of carbohydrate crops by the regulation of sugar partitioning.

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.

In vivo imaging of Ca2+ accumulation during cotton fiber initiation using fluorescent indicator YC3.60.

KEY MESSAGE: Non-tip-focused Ca 2+ gradient indicated by genetically expressing a FRET-based calcium sensor YC3.60 was established in spherical expanding cotton fibers, which is vital for cotton fiber initiation. Cotton fiber is a single cell elongated from ovule epidermis. It is not only the most important natural fiber used in the textile industry but also an ideal model for studying cell differentiation and elongation. Before linear cell growth, cotton fibers undergo spherical expansion at the beginning of initiation. Ca2+, as an important secondary messenger, plays a central role in polarized cell growth including cotton fiber elongation. However, the role of Ca2+ in fiber initiation is far from well understood. In this paper, through ovule culture we demonstrate that Ca2+ is crucial for fiber initiation. Using transgenic cotton expressing the fluorescent Ca2+ indicator YC3.60, we show cellular and intracellular distribution of Ca2+ in cotton ovule epidermis and fiber cells. In the initiating fiber cell, Ca2+ accumulated mainly at the base of the cell, while in the fast elongating cell, the Ca2+ was enriched in the tip region. This cellular distribution of Ca2+ reported by YC3.60 was confirmed by the staining with a Ca2+-sensitive dye fluo-3/AM. Compared to the fluorescent dye staining, the YC3.60 system can reveal more detailed information on the intracellular distribution without photobleaching. Taken together, our data suggest that Ca2+ plays an important role in spherical expansion of cotton fiber initials.

Moderately enhancing cytokinin level by down-regulation of GhCKX expression in cotton concurrently increases fiber and seed yield.

Cotton is the leading natural fiber crop in the world. Cotton seeds are also an important oil and protein source. However, enhancement of fiber abundance usually leads to a smaller seed. Thus, it has become a challenge for cotton breeding to concurrently increase fiber yield and seed yield. To improve cotton yield, we elevated the endogenous cytokinin level in transgenic cotton by constitutive suppression of cytokinin dehydrogenase (CKX), a key negative regulator controlling endogenous cytokinin in plants. The slightly and moderately suppressed transgenic cotton plants showed normal growth and development, while the severely suppressed plants exhibited a typical cytokinin-overproduction alteration. The suppression of CKX led to an enhancement of endogenous cytokinins in transgenic cotton plants. Total cytokinins in moderately suppressed lines, CR-3 and CR-6, increased by 20.4 and 55.5 % respectively, and that in the severely suppressed line (CR-13) increased by 134.2 % compared to the wild type. The moderately suppressed lines showed a delay in leaf senescence, higher photosynthesis, more fruiting branches and bolls, and bigger seed size. Field trials showed that seed yield and lint yield of the moderately suppressed CR-6 line increased by 15.4 and 20.0 %, respectively. Meanwhile, the enhanced cytokinin level in transgenic cottons did not show significant influence on fiber qualities. Our data demonstrated that CKX is a promising gene for crop yield improvement.

Gibberellin overproduction promotes sucrose synthase expression and secondary cell wall deposition in cotton fibers.

Bioactive gibberellins (GAs) comprise an important class of natural plant growth regulators and play essential roles in cotton fiber development. To date, the molecular base of GAs' functions in fiber development is largely unclear. To address this question, the endogenous bioactive GA levels in cotton developing fibers were elevated by specifically up-regulating GA 20-oxidase and suppressing GA 2-oxidase via transgenic methods. Higher GA levels in transgenic cotton fibers significantly increased micronaire values, 1000-fiber weight, cell wall thickness and cellulose contents of mature fibers. Quantitative RT-PCR and biochemical analysis revealed that the transcription of sucrose synthase gene GhSusA1 and sucrose synthase activities were significantly enhanced in GA overproducing transgenic fibers, compared to the wild-type cotton. In addition, exogenous application of bioactive GA could promote GhSusA1 expression in cultured fibers, as well as in cotton hypocotyls. Our results suggested that bioactive GAs promoted secondary cell wall deposition in cotton fibers by enhancing sucrose synthase expression.

Spatiotemporal manipulation of auxin biosynthesis in cotton ovule epidermal cells enhances fiber yield and quality.

The capacity of conventional breeding to simultaneously improve the yield and quality of cotton fiber is limited. The accumulation of the plant hormone indole-3-acetic acid (IAA) in cotton fiber initials prompted us to investigate the effects of genetically engineering increased IAA levels in the ovule epidermis. Targeted expression of the IAA biosynthetic gene iaaM, driven by the promoter of the petunia MADS box gene Floral Binding protein 7 (FBP7), increased IAA levels in the epidermis of cotton ovules at the fiber initiation stage. This substantially increased the number of lint fibers, an effect that was confirmed in a 4-year field trial. The lint percentage of the transgenic cotton, an important component of fiber yield, was consistently higher in our transgenic plants than in nontransgenic controls, resulting in a >15% increase in lint yield. Fiber fineness was also notably improved.

The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters.

Here we report the effect of the 35S promoter sequence on activities of the tissue- and organ-specific gene promoters in tobacco plants. In the absence of the 35S promoter sequence the AAP2 promoter is active only in vascular tissues as indicated by expression of the AAP2:GUS gene. With the 35S promoter sequence in the same T-plasmid, transgenic plants exhibit twofold to fivefold increase in AAP2 promoter activity and the promoter becomes active in all tissue types. Transgenic plants hosting the ovary-specific AGL5:iaaM gene (iaaM coding an auxin biosynthetic gene) showed a wild-type phenotype except production of seedless fruits, whereas plants hosting the AGL5:iaaM gene along with the 35S promoter sequence showed drastic morphological alterations. RT-PCR analysis confirms that the phenotype was caused by activation of the AGL5:iaaM gene in non-ovary organs including roots, stems and flowers. When the pollen-, ovule- and early embryo-specific PAB5:barnase gene (barnase coding a RNase gene) was transformed, the presence of 35S promoter sequence drastically reduced transformation efficiencies. However, the transformation efficiencies were restored in the absence of 35S promoter, indicating that the 35S promoter might activate the expression of PAB5:barnase in non-reproductive organs such as calli and shoot primordia. Furthermore, if the 35S promoter sequence was replaced with the NOS promoter sequence, no alteration in AAP2, AGL5 or PAB5 promoter activities was observed. Our results demonstrate that the 35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter.