Ling-Jian Wang

Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol

We identify a cytochrome P450 gene (CYP6AE14) from cotton bollworm (Helicoverpa armigera), which permits this herbivore to tolerate otherwise inhibitory concentrations of the cotton metabolite, gossypol. CYP6AE14 is highly expressed in the midgut and its expression correlates with larval growth when gossypol is included in the diet. When larvae are fed plant material expressing double-stranded RNA (dsRNA) specific to CYP6AE14, levels of this transcript in the midgut decrease and larval growth is retarded. Both effects are more dramatic in the presence of gossypol. As a glutathione-S-transferase gene (GST1) is silenced in GST1 dsRNA–expressing plants, feeding insects plant material expressing dsRNA may be a general strategy to trigger RNA interference and could find applications in entomological research and field control of insect pests.

Control of Root Cap Formation by MicroRNA-Targeted Auxin Response Factors in Arabidopsis

The plant root cap mediates the direction of root tip growth and protects internal cells. Root cap cells are continuously produced from distal stem cells, and the phytohormone auxin provides position information for root distal organization. Here, we identify the Arabidopsis thaliana auxin response factors ARF10 and ARF16, targeted by microRNA160 (miR160), as the controller of root cap cell formation. The Pro35S:MIR160 plants, in which the expression of ARF10 and ARF16 is repressed, and the arf10-2 arf16-2 double mutants display the same root tip defect, with uncontrolled cell division and blocked cell differentiation in the root distal region and show a tumor-like root apex and loss of gravity-sensing. ARF10 and ARF16 play a role in restricting stem cell niche and promoting columella cell differentiation; although functionally redundant, the two ARFs are indispensable for root cap development, and the auxin signal cannot bypass them to initiate columella cell production. In root, auxin and miR160 regulate the expression of ARF10 and ARF16 genes independently, generating a pattern consistent with root cap development. We further demonstrate that miR160-uncoupled production of ARF16 exerts pleiotropic effects on plant phenotypes, and miR160 plays an essential role in regulating Arabidopsis development and growth.

Control of Plant Trichome Development by a Cotton Fiber MYB Gene

Cotton (Gossypium spp) plants produce seed trichomes (cotton fibers) that are an important commodity worldwide; however, genes controlling cotton fiber development have not been characterized. In Arabidopsis thaliana the MYB gene GLABRA1 (GL1) is a central regulator of trichome development. Here, we show that promoter of a cotton fiber gene, RD22-like1 (RDL1), contains a homeodomain binding L1 box and a MYB binding motif that confer trichome-specific expression in Arabidopsis. A cotton MYB protein GaMYB2/Fiber Factor 1 transactivated the RDL1 promoter both in yeast and in planta. Real-time PCR and in situ analysis showed that GaMYB2 is predominantly expressed early in developing cotton fibers. After transferring into Arabidopsis, GL1::GaMYB2 rescued trichome formation of a gl1 mutant, and interestingly, 35S::GaMYB2 induced seed-trichome production. We further demonstrate that the first intron of both GL1 and GaMYB2 plays a role in patterning trichomes: it acts as an enhancer in trichome and a repressor in nontrichome cells, generating a trichome-specific pattern of MYB gene expression. Disruption of a MYB motif conserved in intron 1 of GL1, WEREWOLF, and GaMYB2 genes affected trichome production. These results suggest that cotton and Arabidopsis use similar transcription factors for regulating trichomes and that GaMYB2 may be a key regulator of cotton fiber development.

Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression.

This work examines the regulation of two sesquiterpene synthases and shows that gibberellin and jasmonate jointly regulate the biosynthesis of sesquiterpenes through the transcription factor MYC2, and the gibberellin signal is transduced to this secondary metabolism pathway through a DELLA–MYC2 interaction Arabidopsis thaliana flowers emit volatile terpenes, which may function in plant–insect interactions. Here, we report that Arabidopsis MYC2, a basic helix-loop-helix transcription factor, directly binds to promoters of the sesquiterpene synthase genes TPS21 and TPS11 and activates their expression. Expression of TPS21 and TPS11 can be induced by the phytohormones gibberellin (GA) and jasmonate (JA), and both inductions require MYC2. The induction of TPS21 and TPS11 results in increased emission of sesquiterpene, especially (E)-β-caryophyllene. DELLAs, the GA signaling repressors, negatively affect sesquiterpene biosynthesis, as the sesquiterpene synthase genes were repressed in plants overaccumulating REPRESSOR OF GA1-3 (RGA), one of the Arabidopsis DELLAs, and upregulated in a penta DELLA-deficient mutant. Yeast two-hybrid and coimmunoprecipitation assays demonstrated that DELLAs, represented by RGA, directly interact with MYC2. In yeast cells, the N terminus of MYC2 was responsible for binding to RGA. MYC2 has been proposed as a major mediator of JA signaling and crosstalk with abscisic acid, ethylene, and light signaling pathways. Our results demonstrate that MYC2 is also connected to GA signaling in regulating a subset of genes. In Arabidopsis inflorescences, it integrates both GA and JA signals into transcriptional regulation of sesquiterpene synthase genes and promotes sesquiterpene production.

Temporal Control of Trichome Distribution by MicroRNA156-Targeted SPL Genes in Arabidopsis thaliana

The microRNA156-targeted SQUAMOSA PROMOTER BINDING PROTEIN LIKE genes, which were reported to define an endogenous phase transition pathway, temporally control the trichome distribution on the stem and inflorescences by activating the trichome negative regulator genes TRICHOMELESS1 and TRIPTYCHON. The production and distribution of plant trichomes is temporally and spatially regulated. After entering into the flowering stage, Arabidopsis thaliana plants have progressively reduced numbers of trichomes on the inflorescence stem, and the floral organs are nearly glabrous. We show here that SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes, which define an endogenous flowering pathway and are targeted by microRNA 156 (miR156), temporally control the trichome distribution during flowering. Plants overexpressing miR156 developed ectopic trichomes on the stem and floral organs. By contrast, plants with elevated levels of SPLs produced fewer trichomes. During plant development, the increase in SPL transcript levels is coordinated with the gradual loss of trichome cells on the stem. The MYB transcription factor genes TRICHOMELESS1 (TCL1) and TRIPTYCHON (TRY) are negative regulators of trichome development. We show that SPL9 directly activates TCL1 and TRY expression through binding to their promoters and that this activation is independent of GLABROUS1 (GL1). The phytohormones cytokinin and gibberellin were reported to induce trichome formation on the stem and inflorescence via the C2H2 transcription factors GIS, GIS2, and ZFP8, which promote GL1 expression. We show that the GIS-dependent pathway does not affect the regulation of TCL1 and TRY by miR156-targeted SPLs, represented by SPL9. These results demonstrate that the miR156-regulated SPLs establish a direct link between developmental programming and trichome distribution.

The Jasmonate-Responsive AP2/ERF Transcription Factors AaERF1 and AaERF2 Positively Regulate Artemisinin Biosynthesis in Artemisia annua L.

Plants of Artemisia annua produce artemisinin, a sesquiterpene lactone widely used in malaria treatment. Amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, and CYP71AV1, a P450 monooxygenase, are two key enzymes of the artemisinin biosynthesis pathway. Accumulation of artemisinin can be induced by the phytohormone jasmonate (JA). Here, we report the characterization of two JA-responsive AP2 family transcription factors--AaERF1 and AaERF2--from A. annua L. Both genes were highly expressed in inflorescences and strongly induced by JA. Yeast one-hybrid and electrophoretic mobility shift assay (EMSA) showed that they were able to bind to the CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs present in both ADS and CYP71AV1 promoters. Transient expression of either AaERF1 or AaERF2 in tobacco induced the promoter activities of ADS or CYP71AV1, and the transgenic A. annua plants overexpressing either transcription factor showed elevated transcript levels of both ADS and CYP71AV1, resulting in increased accumulation of artemisinin and artemisinic acid. By contrast, the contents of these two metabolites were reduced in the RNAi transgenic lines in which expression of AaERF1 or AaERF2 was suppressed. These results demonstrate that AaERF1 and AaERF2 are two positive regulators of artemisinin biosynthesis and are of great value in genetic engineering of artemisinin production.

Gene expression and metabolite profiles of cotton fiber during cell elongation and secondary cell wall synthesis

Cotton fibers elongate rapidly after initiation of elongation, eventually leading to the deposit of a large amount of cellulose. To reveal features of cotton fiber cells at the fast elongation and the secondary cell wall synthesis stages, we compared the respective transcriptomes and metabolite profiles. Comparative analysis of transcriptomes by cDNA array identified 633 genes that were differentially regulated during fiber development. Principal component analysis (PCA) using expressed genes as variables divided fiber samples into four groups, which are diagnostic of developmental stages. Similar grouping results are also found if we use non-polar or polar metabolites as variables for PCA of developing fibers. Auxin signaling, wall-loosening and lipid metabolism are highly active during fiber elongation, whereas cellulose biosynthesis is predominant and many other metabolic pathways are downregulated at the secondary cell wall synthesis stage. Transcript and metabolite profiles and enzyme activities are consistent in demonstrating a specialization process of cotton fiber development toward cellulose synthesis. These data demonstrate that cotton fiber cell at a certain stage has its own unique feature, and developmental stages of cotton fiber cells can be distinguished by their transcript and metabolite profiles. During the secondary cell wall synthesis stage, metabolic pathways are streamed into cellulose synthesis.

Transcriptional regulation of plant secondary metabolism.

Plant secondary metabolites play critical roles in plant-environment interactions. They are synthesized in different organs or tissues at particular developmental stages, and in response to various environmental stimuli, both biotic and abiotic. Accordingly, corresponding genes are regulated at the transcriptional level by multiple transcription factors. Several families of transcription factors have been identified to participate in controlling the biosynthesis and accumulation of secondary metabolites. These regulators integrate internal (often developmental) and external signals, bind to corresponding cis-elements--which are often in the promoter regions--to activate or repress the expression of enzyme-coding genes, and some of them interact with other transcription factors to form a complex. In this review, we summarize recent research in these areas, with an emphasis on newly-identified transcription factors and their functions in metabolism regulation.

Gossypium barbadense genome sequence provides insight into the evolution of extra-long staple fiber and specialized metabolites

Of the two cultivated species of allopolyploid cotton, Gossypium barbadense produces extra-long fibers for the production of superior textiles. We sequenced its genome (AD)2 and performed a comparative analysis. We identified three bursts of retrotransposons from 20 million years ago (Mya) and a genome-wide uneven pseudogenization peak at 11–20 Mya, which likely contributed to genomic divergences. Among the 2,483 genes preferentially expressed in fiber, a cell elongation regulator, PRE1, is strikingly At biased and fiber specific, echoing the A-genome origin of spinnable fiber. The expansion of the PRE members implies a genetic factor that underlies fiber elongation. Mature cotton fiber consists of nearly pure cellulose. G. barbadense and G. hirsutum contain 29 and 30 cellulose synthase (CesA) genes, respectively; whereas most of these genes (>25) are expressed in fiber, genes for secondary cell wall biosynthesis exhibited a delayed and higher degree of up-regulation in G. barbadense compared with G. hirsutum, conferring an extended elongation stage and highly active secondary wall deposition during extra-long fiber development. The rapid diversification of sesquiterpene synthase genes in the gossypol pathway exemplifies the chemical diversity of lineage-specific secondary metabolites. The G. barbadense genome advances our understanding of allopolyploidy, which will help improve cotton fiber quality.

The HD-Zip IV gene GaHOX1 from cotton is a functional homologue of the Arabidopsis GLABRA2

Most of the plant homeodomain-containing proteins play important roles in organ patterning and development, and Arabidopsis GLABRA2 (GL2), a member of the class IV homeodomain-leucine zipper (HD-ZIP) proteins, is a trichome and non-root hair cell regulator. Here we report the analysis of two cotton homeodomain-containing proteins, GaHOX1 and GaHOX2, isolated from the diploid cotton Gossypium arboreum. Both GaHOX1 and GaHOX2 belong to the class IV HD-ZIP family. When expressed under the control of the GL2 promoter, GaHOX1 rescued trichome development of an Arabidopsis glabrous mutant of gl2-2 (SALK_130213), whereas GaHOX2 did not. On the other hand, expression of GaHOX1 with a Cauliflower mosaic virus (CaMV) 35S promoter in the wild-type Arabidopsis plants suppressed the trichome development just as the GL2 ectopic expression. Expression analysis by Northern, RT-PCR and in situ hybridization indicated that GaHOX1 is predominantly expressed in cotton fiber cells at early developmental stages, consistent with its putative role in regulating cotton fiber development, while GaHOX2 is expressed in both fiber and other ovular tissues, including outer and inner integuments. Our results suggest that GaHOX1 is a functional homolog of GL2 in plant trichome development.