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Plant extracellular vesicles (EVs) are membrane-enclosed nanoparticles that play diverse roles in plant development and response. Recently, impressive progress has been made in the isolation and identification of the proteins and RNAs carried in plant EVs; however, the analysis of EV lipid compositions remains rudimentary. Here, we performed lipidomic analysis of Arabidopsis rosette leaf EVs, revealing a high abundance of certain groups of lipids, in particular sphingolipids, in the EVs. Remarkably, the EV sphingolipids are composed of nearly pure glycosylinositolphosphoceramides (GIPCs), which are green lineage abundant and negatively charged. We further showed that the Arabidopsis TETRASPANIN 8 (TET8) knockout mutant has a lower amount of cellular GIPCs and secrets fewer EVs, companied with impaired reactive oxygen species (ROS) burst toward stresses. Exogenous application of GIPCs promoted the secretion of EVs and ROS burst in both the WT and tet8 mutant. The characteristic enrichment of sphingolipid GIPCs provides valuable insights into the biogenesis and function of plant EVs.

Summary Almost all plants form trichomes, which protect them against insect herbivores by forming a physical barrier and releasing chemical repellents. Glandular trichomes produce a variety of specialized defensive metabolites, including volatile terpenes. Previous studies have shown that the defence hormone jasmonic acid (JA) affects trichome development and induces terpene synthases (TPSs) but the underlying molecular mechanisms remain unclear. Here, we characterized a loss‐of‐function allele of the HD‐ZIP IV transcription factor woolly (wo) and analysed its role in mediating JA signalling in tomato. We showed that knockout of wo led to extensive trichome defects, including structural and functional changes in type VI glandular trichomes, and a dramatic reduction in terpene levels. We further found that wo directly binds to TPS gene promoters to recruit SlMYC1, a JA signalling modulator, and that together these transcription factors promote terpene biosynthesis in tomato trichomes. The wo/SlMYC1 regulatory module is inhibited by SlJAZ2 through a competitive binding mechanism, resulting in a fine‐tuned JA response in tomato trichomes. Enhanced expression of SlMYC1 substantially increased terpene levels and improved tomato resistance to spider mites. Interestingly, we also found that SlMYC1 plays an additional role in glandular cell division and expansion in type VI trichomes, independent of JA. Together, our results reveal a novel, JA‐mediated regulatory mechanism that promotes insect resistance in tomato.

The reactive electrophilic species (RES), typically the molecules bearing α,β-unsaturated carbonyl group, are widespread in living organisms and notoriously known for their damaging effects. Many of the mycotoxins released from phytopathogenic fungi are RES and their contamination to cereals threatens food safety worldwide. However, due to their high reactivity, RES are also used by host organisms to synthesize specific metabolites. The evolutionary conserved glyoxalase (GLX) system scavenges the cytotoxic α-oxoaldehydes that bear RES groups, which cause host disorders and diseases. In cotton, a specialized enzyme derived from glyoxalase I (GLXI) through gene duplications and named as specialized GLXI (SPG), acts as a distinct type of aromatase in the gossypol pathway to transform the RES intermediates into the phenolic products. In this review, we briefly introduce the research progress in understanding the RES, especially the RES-type mycotoxins, the GLX system and SPG, and discuss their application potential in detoxification and synthetic biology.

The exquisite chemodiversity of terpenoids is the product of the large diverse terpene synthase (TPS) superfamily. Here, by using structural and phylogenetic analyses and site-directed mutagenesis, we identified a residue (Cys440 in Nicotiana tabacum 5-epi-aristolochene synthase) proximal to an ion-binding motif common to all TPSs and named the preNSE/DTE residue, which determines the product specificity of sesquiterpene synthases from different plant species. In sesquiterpene synthases catalyzing 1,10-cyclization (1,10-cyclases) of farnesyl diphosphate, mutation of the residue in both specific and promiscuous 1,10-cyclases from different lineages leads to the accumulation of monocyclic germacrene A-11-ol, which is "short-circuited" from complex cyclization cascades, suggesting a key role of this residue in generating the first common intermediate of 1,10-cyclization. Altering this residue in a specific 1,11-cyclase results in alternative 1,10-cyclization products. Moreover, the preNSE/DTE residue can be harnessed to engineer highly specific sesquiterpene synthases for an improved proportion of high-value terpenoids, such as patchoulol, a main constituent of several traditional Chinese medicines that could treat SARS-CoV-2.

Sphingolipids, which comprise membrane systems together with other lipids, are ubiquitous in cellular organisms. They show a high degree of diversity across plant species and vary in their structures, properties, and functions. Benefiting from the development of lipidomic techniques, over 300 plant sphingolipids have been identified. Generally divided into free long-chain bases (LCBs), ceramides, glycosylceramides (GlcCers) and glycosyl inositol phosphoceramides (GIPCs), plant sphingolipids exhibit organized aggregation within lipid membranes to form raft domains with sterols. Accumulating evidence has revealed that sphingolipids obey certain trafficking and distribution rules and confer unique properties to membranes. Functional studies using sphingolipid biosynthetic mutants demonstrate that sphingolipids participate in plant developmental regulation, stimulus sensing, and stress responses. Here, we present an updated metabolism/degradation map and summarize the structures of plant sphingolipids, review recent progress in understanding the functions of sphingolipids in plant development and stress responses, and review sphingolipid distribution and trafficking in plant cells. We also highlight some important challenges and issues that we may face during the process of studying sphingolipids., This review summarizes recent progress in plant sphingolipid metabolism, diversity, distribution, trafficking, and function. The intersections among these topics are also discussed, then current challenges and issues in the study of plant sphingolipids are highlighted.

BackgroundCotton fiber is a model system for studying plant cell development. At present, the functions of many transcription factors in cotton fiber development have been elucidated, however, the roles of auxin response factor (ARF) genes in cotton fiber development need be further explored.ResultsHere, we identify auxin response factor (ARF) genes in three cotton species: the tetraploid upland cottonG. hirsutum, which has 73 ARF genes, and its putative extent parental diploidsG. arboreumandG. raimondii, which have 36 and 35 ARFs, respectively. Ka and Ks analyses revealed that inG. hirsutum ARFgenes have undergone asymmetric evolution in the two subgenomes. The cotton ARFs can be classified into four phylogenetic clades and are actively expressed in young tissues. We demonstrate thatGhARF2b, a homolog of the ArabidopsisAtARF2, was preferentially expressed in developing ovules and fibers. Overexpression ofGhARF2bby a fiber specific promoter inhibited fiber cell elongation but promoted initiation and, conversely, its downregulation by RNAi resulted in fewer but longer fiber. We show that GhARF2b directly interacts with GhHOX3 and represses the transcriptional activity of GhHOX3 on target genes.ConclusionOur results uncover an important role of the ARF factor in modulating cotton fiber development at the early stage.

Background Cotton (Gossypium spp.) is the most important world-wide fiber crop but salt stress limits cotton production in coastal and other areas. Growth regulation factors (GRFs) play regulatory roles in response to salt stress, but their roles have not been studied in cotton under salt stress. Results We identified 19 GRF genes in G. raimondii, 18 in G. arboreum, 34 in G. hirsutum and 45 in G. barbadense, respectively. These GRF genes were phylogenetically analyzed leading to the recognition of seven GRF clades. GRF genes from diploid cottons (G. raimondii and G. arboreum) were largely retained in allopolyploid cotton, with subsequent gene expansion in G. barbadense relative to G. hirsutum. Most G. hirsutum GRF (GhGRF) genes are preferentially expressed in young and growing tissues. To explore their possible role in salt stress, we used qRT-PCR to study expression responses to NaCl treatment, showing that five GhGRF genes were down-regulated in leaves. RNA-seq experiments showed that seven GhGRF genes exhibited decreased expression in leaves under NaCl treatment, three of which (GhGRF3, GhGRF4, and GhGRF16) were identified by both RNA-seq and qRT-PCR. We also identified six and three GRF genes that exhibit decreased expression under salt stress in G. arboreum and G. barbadense, respectively. Consistent with its lack of leaf withering or yellowing under the salt treatment conditions, G. arboreum had better salt tolerance than G. hirsutum and G. barbadense. Our results suggest that GRF genes are involved in salt stress responses in Gossypium. Conclusion In summary, we identified candidate GRF genes that were involved in salt stress responses in cotton.

Plant cell growth involves a complex interplay among cell-wall expansion, biosynthesis, and, in specific tissues, secondary cell wall (SCW) deposition, yet the coordination of these processes remains elusive. Cotton fiber cells are developmentally synchronous, highly elongated, and contain nearly pure cellulose when mature. Here, we report that the transcription factor GhTCP4 plays an important role in balancing cotton fiber cell elongation and wall synthesis. During fiber development the expression of miR319 declines while GhTCP4 transcript levels increase, with high levels of the latter promoting SCW deposition. GhTCP4 interacts with a homeobox-containing factor, GhHOX3, and repressing its transcriptional activity. GhTCP4 and GhHOX3 function antagonistically to regulate cell elongation, thereby establishing temporal control of fiber cell transition to the SCW stage. We found that overexpression of GhTCP4A upregulated and accelerated activation of the SCW biosynthetic pathway in fiber cells, as revealed by transcriptome and promoter activity analyses, resulting in shorter fibers with varied lengths and thicker walls. In contrast, GhTCP4 downregulation led to slightly longer fibers and thinner cell walls. The GhHOX3–GhTCP4 complex may represent a general mechanism of cellular development in plants since both are conserved factors in many species, thus providing us a potential molecular tool for the design of fiber traits.

Summary Cotton cultivars have evolved to produce extensive, long, seed‐born fibers important for the textile industry, but we know little about the molecular mechanism underlying spinnable fiber formation. Here, we report how PACLOBUTRAZOL RESISTANCE 1 (PRE1) in cotton, which encodes a basic helix‐loop‐helix (bHLH) transcription factor, is a target gene of spinnable fiber evolution.Differential expression of homoeologous genes in polyploids is thought to be important to plant adaptation and novel phenotypes. PRE1 expression is specific to cotton fiber cells, upregulated during their rapid elongation stage and A‐homoeologous biased in allotetraploid cultivars. Transgenic studies demonstrated that PRE1 is a positive regulator of fiber elongation.We determined that the natural variation of the canonical TATA‐box, a regulatory element commonly found in many eukaryotic core promoters, is necessary for subgenome‐biased PRE1 expression, representing a mechanism underlying the selection of homoeologous genes.Thus, variations in the promoter of the cell elongation regulator gene PRE1 have contributed to spinnable fiber formation in cotton. Overexpression of GhPRE1 in transgenic cotton yields longer fibers with improved quality parameters, indicating that this bHLH gene is useful for improving cotton fiber quality.

Summary Plant engineered to express double-stranded RNA (dsRNA) can target the herbivorous insect gene for silencing. Although mounting evidence has emerged to support feasibility of this new pest control technology, field application is slow largely due to lack of potent targets. Here, we show that suppression of the gene encoding NDUFV2, a subunit of mitochondrial complex I that catalyses NADH dehydrogenation in respiratory chain, was highly lethal to insects. Feeding cotton bollworm (Helicoverpa armigera) larvae with transgenic cotton tissues expressing NDUFV2 dsRNA led to mortality up to 80% within 5 days, and almost no larvae survived after 7 days of feeding, due to the altered mitochondrial structure and activity. Transcriptome comparisons showed a drastic repression of dopa decarboxylase genes. Reciprocal assays with Asian corn borer (Ostrinia furnacalis), another lepidopteran species, revealed the sequence-specific effect of NDUFV2 suppression. Furthermore, the hemipteran lugus Apolygus lucorum was also liable to NDUFV2 repression. These data demonstrate that the mitochondrial complex I is a promising target with both sequence specificity and wide applicability for the development of new-generation insect-proof crops.

The plant extracellular vesicles (EVs) are lipid-enveloped nano-particles containing proteins, nucleic acids and metabolites and function in plant development and response. The Arabidopsis four transmembrane protein TETRASPANIN 8 (TET8) knock-out mutant tet8 secreted less EVs than the wild-type (WT). In this report, we show that the tet8 mutant was attenuated in the plant hormone jasmonate (JA) accumulation in response to mechanical wounding treatment. We also noticed that the EVs contained a high level of phospholipids phosphatidic acids (PAs) which may serve as precursors of JA biosynthesis during wound-triggered-self-healing processes. Thus, we propose an open question about a potential role of EVs or TET8 or both in damage-associated JA response.

Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto-δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion.

We isolated a 2-alkenal reductase (AaAER) gene from Artemisia annua L. cDNA library through homologous cloning strategy. Enzymatic properties of AaAER, including substrate selectivity, enzyme kinetics, and key factors affecting enzyme activity, were characterized in vitro. We found that AaAER mainly functions in catalyzing the reduction of adjacent straight chain “C = C” double bond in the carbonyl group. This suggests the possibility of its participation in downstream events of the HPL (hydroperoxide lyase) pathway and contribution to cell detoxification in plants. Although, AaAER shares some substrates with AaDbr 1 and AaDbr 2, which are involved in artemisinin biosynthesis, AaAER was unable to reduce the adjacent double bond of carbonyl group in artemisinic aldehyde. We also analyzed the possible causes for highly homologous reductases with different types of substrates and non-homologous enzymes sharing the same substrate. The identification of a novel double bond reductase in A. annua provides an opportunity to explore the mechanism underlying the detoxification of aldehyde ketone molecules, which are generated by HPL downstream pathway and the redox process in the metabolic pathway of artemisinin synthesis.

Plant secondary metabolites and their biosynthesis have attracted great interest, but investigations of the activities of hidden intermediates remain rare. Gossypol and related sesquiterpenes are the major phytoalexins in cotton. Among the six biosynthetic intermediates recently identified, 8-hydroxy-7-keto-δ-cadinene (C234) crippled the plant disease resistance when accumulated upon gene silencing. C234 harbours an α,β-unsaturated carbonyl thus is a reactive electrophile species. Here, we show that C234 application also dampened the Arabidopsis resistance against the bacterial pathogen Pseudomonas syringae pv. maculicola ( Psm ). We treated Arabidopsis with C234, Psm and ( Psm +C234), and analysed the leaf transcriptomes. While C234 alone exerted a mild effect, it greatly stimulated an over-response to the pathogen. Of the 7335 genes affected in the ( Psm +C234)-treated leaves, 3476 were unresponsive without the chemical, in which such functional categories as ‘nucleotides transport’, ‘vesicle transport’, ‘MAP kinases’, ‘G-proteins’, ‘protein assembly and cofactor ligation’ and ‘light reaction’ were enriched, suggesting that C234 disturbed certain physiological processes and the protein complex assembly, leading to distorted defence response and decreased disease resistance. As C234 is efficiently metabolized by CYP71BE79, plants of cotton lineage have evolved a highly active enzyme to prevent the phytotoxic intermediate accumulation during gossypol pathway evolution. This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management’.

As the most diverse natural organic compounds, plant secondary metabolites play critical roles in regulating plant growth and development, facilitating environmental adaptation and defending against insects and pathogens. Terpenoids constitute one of the largest families of secondary metabolites, and have important physiological and ecological functions. Some of them even show great pharmaceutical values. In recent decades, great achievements have been made in isolation and characterization of terpenoids, elucidation of their biosynthetic pathways and regulation mechanisms, as well as genetic engineering. This review discusses recent progress in terpenoid metabolism and regulation.

Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.

Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665–673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects. Electronic supplementary material The online version of this article (doi:10.1007/s11103-013-0030-7) contains supplementary material, which is available to authorized users.

Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5 and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with camphene and 1,8-cineole as major products, respectively, and AaTPS2 produced a single product, β-myrcene. Although both Mg2+ and Mn2+ were able to support their catalytic activities, altered product spectrum was observed in the presence of Mn2+ for AaTPS2 and AaTPS5. Analysis of extracts of aerial tissues and root of A. annua with gas chromatography-mass spectrometry (GC-MS) detected more than 20 monoterpenes, of which the three enzymes constituted more than 1/3 of the total. Mechanical wounding induced the expression of all three monoterpene synthase genes, and transcript levels of AaTPS5 and AaTPS6 were also elevated after treatments with phytohormones of methyl jasmonate (MeJA), salicylic acid (SA) and gibberellin (GA), suggesting a role of these monoterpene synthases in plant-environment interactions. The three new monoterpene synthases reported here further our understanding of molecular basis of monoterpene biosynthesis and regulation in plant.

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.

Cotton fiber is proposed to share some similarity with the Arabidopsis thaliana leaf trichome, which is regulated by the MYB-bHLH-WD40 transcription complex. Although several MYB transcription factors and WD40 family proteins in cotton have been characterized, little is known about the role of bHLH family proteins in cotton. Here, we report that GhDEL65, a bHLH protein from cotton (Gossypium hirsutum), is a functional homologue of Arabidopsis GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) in regulating trichome development. Transcripts of GhDEL65 were detected in 0 ∼ 1 days post-anthesis (DPA) ovules and abundant in 3-DPA fibers, implying that GhDEL65 may act in early fiber development. Ectopic expression of GhDEL65 in Arabidopsis gl3 egl3 double mutant partly rescued the trichome development, and constitutive expression of GhDEL65 in wild-type plants led to increased trichome density on rosette leaves and stems, mainly by activating the transcription of two key positive regulators of trichome development, GLABRA1 (GL1) and GLABRA2 (GL2), and suppressed the expression of a R3 single-repeat MYB factor TRIPTYCHON (TRY). GhDEL65 could interact with cotton R2R3 MYB transcription factors GhMYB2 and GhMYB3, as well as the WD40 protein GhTTG3, suggesting that the MYB-bHLH-WD40 protein complex also exists in cotton fiber cell, though its function in cotton fiber development awaits further investigation.

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.

Artemisinin, an effective drug against Plasmodium species that cause malaria, is a sesquiterpene lactone isolated from Artemisia annua. The yield of artemisinin in plants is relatively low, and it is influenced by various environmental growing conditions. In Arabidopsis, cryptochrome 1 (CRY1) is one of the key receptors that perceive light signals, and its overexpression promotes accumulation of secondary metabolites. In this study, overexpression of Arabidopsis CRY1 in A. annua resulted in increased accumulation of both artemisinin and anthocyanins. Under blue light, transgenic plants expressing high level of AtCRY1 showed a variety of blue light-induced responses, including inhibition of hypocotyl elongation and cotyledon expansion, dwarfism, and purpuring of aerial organs. Reverse transcription polymerase chain reaction analysis revealed that expression levels of the phenylalanine ammonia-lyase- and chalcone synthase-encoding genes were elevated and that anthocyanin accumulation was increased. Expression analysis of genes encoding farnesyl diphosphate synthase (FPS), amorpha-4,11-diene synthase (ADS), and CYP71AV1, three important enzymes in artemisinin biosynthesis, indicated that transcript abundance of all three genes, FPS, ADS, and CYP71AV1, increased in AtCRY1 plants. Based on high performance liquid chromatography analysis, artemisinin content in these plants increased by 30∼40%, when compared to control. These results have demonstrated that alteration of light signaling components could provide a viable approach for enhancing production of the secondary metabolite artemisinin.

During leaf organogenesis, a critical step for normal leaf primordium initiation is the repression of the class 1 KNOTTED1-like homeobox (KNOX) genes. After leaf primordia are formed, they must establish polarity for normal leaf morphogenesis. Recent studies have led to the identification of a number of genes that participate in the class 1 KNOX gene repression and/or the leaf polarity establishment. ASTMMETRIC LEAVES1 and 2 (AS1 and AS2) are two of these genes, which are critical for both of these two processes. As a first step towards understanding the molecular genetic basis of the AS1-AS2 action, we determined the subcellular localizations of the two proteins in both tobacco BY2 cells and Arabidopsis plants, by fusing them to yellow/cyan fluorescent protein (YFP/CFP). Our data showed that AS1 and AS2 alone were predominantly localized in the nucleolus and the nucleoplasm, respectively. The presence of both AS1 and AS2 proteins in the same interphase cell demonstrated their co-localization in both nucleolus and nucleoplasm. In addition, AS1 alone was able to associate with the condensed chromosome in the metaphase cell. Our data suggest that AS1, AS2 and the AS1-AS2 protein complex may have distinct functions, which are all required for normal plant development.

S-adenosyl-l-homocysteine hydrolase (SAHH) is a key enzyme for maintenance of cellular transmethylation potential. Although a cytokinin-binding activity had been hypothesized for SAHH, the relation between cytokinin and transmethylation reactions has not been elucidated. Here we show that, of the two Arabidopsis thaliana SAHH genes, AtSAHH1 has a much higher expression level than AtSAHH2. A T-DNA insertion mutant of AtSAHH1 (sahh1-1) and the RNA interference (RNAi) plants (dsAtSAHH2) accumulated a higher level of cytokinins, exhibited phenotypic changes similar to those of cytokinin-overproducers, and their global DNA methylation status was reduced. On the other hand, cytokinins positively regulate the transmethylation pathway genes, including AtSAHH1, AtADK1 (for adenosine kinase), and this regulation involves the cytokinin activity. Furthermore, expression of three cytosine DNA methyltransferase genes examined was inducible by cytokinin treatment. Unlike adenine and adenosine which are SAHH inhibitors, the adenine-type cytokinins have no effect on SAHH activity at protein level. Changing of endogenous cytokinin levels by transgene expression resulted in alterations of DNA methylation status in the sahh1-1 background, suggesting that cytokinins promote DNA methylation, at least under transmethylation stringent conditions. These data demonstrate that the phytohormone cytokinin plays a role in promoting transmethylation reactions, including DNA methylation.

Cuticle, including wax and cutin, is the barrier covering plant aerial organs and protecting the inner tissues. The Arabidopsis thaliana ATP-binding cassette (ABC) transporter CER5 (AtWBC12) has been identified as a wax exporter. In agreement with the latest report of another wax exporter, AtWBC11, here we show that atwbc11 mutants displayed organ fusions and stunted growth, and became vulnerable to chlorophyll leaching and toluidine blue staining. Chemical analysis showed that wax and cutin monomers were both reduced in the atwbc11 mutant. AtWBC11 was widely expressed in aerial organs. Interestingly, we found that the expression was light dependent, and the phytohormone ABA up-regulated AtWBC11 expression. We also found that while the AtWBC11 promoter had a broad pattern of activity, the expression was converted to epidermis specific when the reporter gene was fused to AtWBC11 cDNA. Furthermore, RNA blot analysis supported epidermis-specific expression of AtWBC11. Our results support that AtWBC11 is involved in cuticle development.

Of the homeodomain protein superfamily homeodomain-leucine zipper (HD-ZIP) proteins form a plant-specific subfamily. A number of HD-ZIP transcription factors have been reported to play important roles in various aspects of plant growth and development, however, functions of most members remain unknown. Here, we report the characterization of an Arabidopsis semidominant mutant, upcurved leaf1 ( ucl1 ). Besides having upcurved leaves, the homozygous mutant plants are male sterile due to anther non-dehiscence. Molecular and genetic analysis indicated that the phenotypes of ucl1 were resulted from increased expression of At2g32370 , which encodes a member of class IV HD-ZIP protein, HDG3. We isolated the full-length cDNA of HDG3 and found a splicing site different from the annotated information. RT-PCR analysis indicated that HDG3 is specifically expressed in flowers and the promoter-GUS analysis revealed that it is only expressed in anthers. In homozygous ucl1 anthers, the expression levels of three positive regulators of anther dehiscence, MYB26 , NST1 and NST2 were down-regulated. Taken together, our data demonstrate that HDG3 plays a negative role in role in regulation of anther dehiscence.

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.

Aerial surfaces of most land plants have epidermal hairs (trichomes). Both Arabidopsis thaliana trichomes and cotton fibers (seed hairs) are nonglandular hairs with unicellular structure. Here we outline the recent progress on molecular dissection of Arabidopsis trichome development, including the transcription factors regulating trichome initiation, phytohormones influencing trichome density, and the cytoskeleton formation system governing trichome morphogenesis. There is increasing evidence showing that these processes also control cotton fiber production. As several cotton fiber genes are able to rescue the Arabidopsis trichome mutants, we discuss the conservation between Arabidopsis trichome and cotton fiber development.

Terpenoids serve as both constitutive and inducible defense chemicals in many plant species, and volatile terpenes participate in plant a indirect defense by attracting natural enemies of the herbivores. The rice (Oryza sativa L.) genome contains about 50 genes encoding putative terpene synthases (TPSs). Here we report that two of the rice sesquiterpene synthase genes, OsTPS3 and OsTPS13, encode (E)-beta-caryophyllene synthase and (E,E)-farnesol synthase, respectively. In vitro, the recombinant protein of OsTPS3 catalyzed formation of (E)-beta-caryophyllene and several other sesquiterpenes, including beta-elemene and alpha-humulene, all being components of inducible volatiles of rice plants. The transcript levels of OsTPS3 exhibit a circadian rhythm of fluctuation, and its expression was also greatly induced by methyl jasmonate (MeJA). In addition, expression of OsTPS3 in transgenic plants of Arabidopsis thaliana resulted in emitting high quantities of OsTPS3 products. We also overexpressed OsTPS3 in rice plants which then produced more (E)-beta-caryophyllene after MeJA treatment. Finally, we found that the MeJA-treated transgenic rice plants attracted more parasitoid wasps of Anagrus nilaparvatae than the wild-type. These results demonstrate that OsTPS3, an enzyme catalyzing the formation of volatile sesquiterpenes, plays a role in indirect defense of rice plants.

Among plant secondary metabolites terpenoids are a structurally most diverse group; they function as phytoalex- ins in plant direct defense, or as signals in indirect defense responses which involves herbivores and their natural enemies. In recent years, more and more attention has been paid to the investigation of the ecological role of plant terpenoids. The biosynthesis pathways of monoterpenes, sesquiterpenes, and diterpenes include the synthesis of C5 precursor isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), the synthe- sis of the immediate diphosphate precursors, and the formation of the diverse terpenoids. Terpene synthases (TPSs) play a key role in volatile terpene synthesis. By expression of the TPS genes, significant achievements have been made on metabolic engineering to increase terpenoid production. This review mainly summarizes the recent research progress in elucidating the ecological role of terpenoids and characterization of the enzymes involved in the terpenoid biosynthesis. Spatial and temporal regulations of terpenoids metabolism are also discussed.

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.

Immunity deteriorates with age in animals but comparatively little is known about the temporal regulation of plant resistance to herbivores. The phytohormone jasmonate (JA) is a key regulator of plant insect defense. Here, we show that the JA response decays progressively in Arabidopsis. We show that this decay is regulated by the miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9) group of proteins, which can interact with JA ZIM-domain (JAZ) proteins, including JAZ3. As SPL9 levels gradually increase, JAZ3 accumulates and the JA response is attenuated. We provide evidence that this pathway contributes to insect resistance in young plants. Interestingly however, despite the decay in JA response, older plants are still comparatively more resistant to both the lepidopteran generalist Helicoverpa armigera and the specialist Plutella xylostella, along with increased accumulation of glucosinolates. We propose a model whereby constitutive accumulation of defense compounds plays a role in compensating for age-related JA-response attenuation during plant maturation., Immunity deteriorates with age in animals but less is known about how aging affects immunity in plants. Here, Mao et al. show that responsiveness to the major insect defense hormone JA declines as plants age, but insect resistance still increases as plants accumulate secondary metabolites such as glucosinolates.

Cotton fibres are unusually long, single-celled epidermal seed trichomes and a model for plant cell growth, but little is known about the regulation of fibre cell elongation. Here we report that a homeodomain-leucine zipper (HD-ZIP) transcription factor, GhHOX3, controls cotton fibre elongation. GhHOX3 genes are localized to the 12th homoeologous chromosome set of allotetraploid cotton cultivars, associated with quantitative trait loci (QTLs) for fibre length. Silencing of GhHOX3 greatly reduces (>80%) fibre length, whereas its overexpression leads to longer fibre. Combined transcriptomic and biochemical analyses identify target genes of GhHOX3 that also contain the L1-box cis-element, including two cell wall loosening protein genes GhRDL1 and GhEXPA1. GhHOX3 interacts with GhHD1, another homeodomain protein, resulting in enhanced transcriptional activity, and with cotton DELLA, GhSLR1, repressor of the growth hormone gibberellin (GA). GhSLR1 interferes with the GhHOX3–GhHD1 interaction and represses target gene transcription. Our results uncover a novel mechanism whereby a homeodomain protein transduces GA signal to promote fibre cell elongation.

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.

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.

Plant metabolites vary at different stages of their life cycle. Although it is well documented that environmental factors stimulate biosynthesis of secondary metabolites, the regulation by endogenous developmental cues remains poorly understood. The microRNA156 (miR156)-targeted squamosa promoter binding protein-like (SPL) factors function as a major age cue in regulating developmental phase transition and flowering. We show here that the miR156-targeted SPL transcription factor plays an important role in the spatiotemporal regulation of sesquiterpene biosynthesis. In Arabidopsis thaliana, the miR156-SPL module regulates the formation of (E)-β-caryophyllene in the flowering stage through modulating expression of the sesquiterpene synthase gene TPS21. We demonstrated that SPL9 directly binds to TPS21 promoter and activates its expression. In the perennial fragrant herb Pogostemon cablin, the accumulation of patchouli oil, largely composed of sesquiterpenes dominated by (-)-patchoulol, is also age-regulated, and the SPL promotes biosynthesis of sesquiterpenes in elder plants by upregulating patchoulol synthase (PatPTS) gene expression. As miR156-SPLs are highly conserved in plants, our finding not only uncovers a molecular link between developmental timing and sesquiterpene production but also suggests a new strategy to engineer plants for accelerated growth with enhanced production of terpenoids.

The relationship among transfer and expression of auxin binding protein gene (abp), auxin (NAA)-induced plasmalemma hyperpolarity and sensibility to auxin during protoplast culture was studied by measuring transmembrane potential difference (Em) and culturing the protoplasts of sense and antisenseabp transgenic tobacco. The concentration of NAA inducing the highest degree of hyperpolarity of senseabp transgenic tobacco protoplasts was lower than the control, and in protoplast culture, their sensibility to auxin increased. The concentration of antisenseabp transgenic tobacco protoplasts was higher than the control, and in protoplast culture, their sensibility to auxin decreased. These results demonstrated that ABP synthesized in endoplasmic reticulum needed to transport to cell membrane and functioned there.

The miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factors function as an endogenous age cue in regulating plant phase transition and phase-dependent morphogenesis, but the control of SPL output remains poorly understood. In Arabidopsis thaliana the spatial pattern of trichome is a hallmark of phase transition and governed by SPLs. Here, by dissecting the regulatory network controlling trichome formation on stem, we show that the miR171-targeted LOST MERISTEMS 1 (LOM1), LOM2 and LOM3, encoding GRAS family members previously known to maintain meristem cell polarity, are involved in regulating the SPL activity. Reduced LOM abundance by overexpression of miR171 led to decreased trichome density on stems and floral organs, and conversely, constitutive expression of the miR171-resistant LOM (rLOM) genes promoted trichome production, indicating that LOMs enhance trichome initiation at reproductive stage. Genetic analysis demonstrated LOMs shaping trichome distribution is dependent on SPLs, which positively regulate trichome repressor genes TRICHOMELESS 1 (TCL1) and TRIPTYCHON (TRY). Physical interaction between the N-terminus of LOMs and SPLs underpins the repression of SPL activity. Importantly, other growth and developmental events, such as flowering, are also modulated by LOM-SPL interaction, indicating a broad effect of the LOM-SPL interplay. Furthermore, we provide evidence that MIR171 gene expression is regulated by its targeted LOMs, forming a homeostatic feedback loop. Our data uncover an antagonistic interplay between the two timing miRNAs in controlling plant growth, phase transition and morphogenesis through direct interaction of their targets., Author Summary MicroRNAs are important aging regulators in many organisms. In Arabidopsis the miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factors play important roles as an endogenous age cue in programming phase transition and phase-dependent morphogenesis, including trichome patterning. However, how the timely increasing SPL output is modulated remains elusive. By dissecting the regulatory network controlling trichome formation on stem, we show that a group of GRAS family members, LOST MERISTEMS 1 (LOM1), LOM2 and LOM3, targeted by timing miR171, function in modulating the SPL activity through direct protein-protein interaction. LOMs promote trichome formation through attenuating the SPL (such as SPL9) activity of trichome repression. The LOM-SPL interaction affects many aspects of plant growth and development, including flowering, aging and chlorophyll biosynthesis. Interestingly, MIR171A gene expression is regulated by its own targets (LOMs), forming a feedback loop to program plant life. Our study establishes an age-dependent regulatory network composed of two timing miRNAs which act oppositely through direct interaction of their target proteins.

Most TPSs (terpene synthases) contain plasticity residues that are responsible for diversified terpene products and functional evolution, which provide a potential for improving catalytic efficiency. Artemisinin, a sesquiterpene lactone from Artemisia annua L., is widely used for malaria treatment and progress has been made in engineering the production of artemisinin or its precursors. In the present paper, we report a new sesquiterpene synthase from A. annua, AaBOS (A. annua α-bisabolol synthase), which has high sequence identity with AaADS (A. annua amorpha-4,11-diene synthase), a key enzyme in artemisinin biosynthesis. Comparative analysis of the two enzymes by domain-swapping and structure-based mutagenesis led to the identification of several plasticity residues, whose alteration changed the product profile of AaBOS to include γ-humulene as the major product. To elucidate the underlying mechanisms, we solved the crystal structures of AaBOS and a γ-humulene-producing AaBOS mutant (termed AaBOS-M2). Among the plasticity residues, position 399, located in the substrate-binding pocket, is crucial for both enzymes. In AaBOS, substitution of threonine for leucine (AaBOSL339T) is required for γ-humulene production; whereas in AaADS, replacing the threonine residue with serine (AaADST399S) resulted in a substantial increase in the activity of amorpha-4,11-diene production, probably as a result of accelerated product release. The present study demonstrates that substitution of plasticity residues has potential for improving catalytic efficiency of the enzyme.

Plant growth requires cell wall extension. The cotton AtRD22-Like 1 gene GhRDL1, predominately expressed in elongating fiber cells, encodes a BURP domain-containing protein. Here, we show that GhRDL1 is localized in cell wall and interacts with GhEXPA1, an α-expansin functioning in wall loosening. Transgenic cotton overexpressing GhRDL1 showed an increase in fiber length and seed mass, and an enlargement of endopleura cells of ovules. Expression of either GhRDL1 or GhEXPA1 alone in Arabidopsis led to a substantial increase in seed size; interestingly, their co-expression resulted in the increased number of siliques, the nearly doubled seed mass, and the enhanced biomass production. Cotton plants overexpressing GhRDL1 and GhEXPA1 proteins produced strikingly more fruits (bolls), leading to up to 40% higher fiber yield per plant without adverse effects on fiber quality and vegetative growth. We demonstrate that engineering cell wall protein partners has a great potential in promoting plant growth and crop yield.

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.

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.

After germination, plants enter juvenile vegetative phase and then transition to an adult vegetative phase before producing reproductive structures. The character and timing of the juvenile-to-adult transition vary widely between species. In annual plants, this transition occurs soon after germination and usually involves relatively minor morphological changes, whereas in trees and other perennial woody plants it occurs after months or years and can involve major changes in shoot architecture. Whether this transition is controlled by the same mechanism in annual and perennial plants is unknown. In the annual forb Arabidopsis thaliana and in maize (Zea mays), vegetative phase change is controlled by the sequential activity of microRNAs miR156 and miR172. miR156 is highly abundant in seedlings and decreases during the juvenile-to-adult transition, while miR172 has an opposite expression pattern. We observed similar changes in the expression of these genes in woody species with highly differentiated, well-characterized juvenile and adult phases (Acacia confusa, Acacia colei, Eucalyptus globulus, Hedera helix, Quercus acutissima), as well as in the tree Populus x canadensis, where vegetative phase change is marked by relatively minor changes in leaf morphology and internode length. Overexpression of miR156 in transgenic P. x canadensis reduced the expression of miR156-targeted SPL genes and miR172, and it drastically prolonged the juvenile phase. Our results indicate that miR156 is an evolutionarily conserved regulator of vegetative phase change in both annual herbaceous plants and perennial trees., Author Summary The existence of discrete juvenile and adult phases of vegetative development in plants was first recognized in trees, in which these phases are usually prolonged and quite stable. Annual plants also undergo changes in vegetative morphology during shoot development, but the relationship between this process and vegetative phase change in trees is unclear. This is because both the timing and the nature of the morphological changes that mark these transitions are different in these groups of plants. Here we show that the expression pattern of miR156—a master regulator of vegetative phase change in Arabidopsis and maize—is conserved in woody plants with well-defined juvenile and adult phases, and we show that over-expression of this microRNA prolongs the expression of the juvenile phase in the tree Populus x canadensis. Our results indicate that the mechanism of the juvenile-to-adult transition is likely conserved throughout flowering plants.

Cotton (Gossypium spp.) plants produce seed trichomes that are the most important textile fiber. Fiber cell initiation and elongation are two key developmental stages that determine the final quality of fiber. A large number of genes have been isolated by transcriptome analysis of these two stages. Here we sum up recent research progress in functional identification of cotton fiber genes, with emphasis on transcription factors and phytohormone signaling pathways, and the fiber-specific or fiber-active promoters.

Cytochrome P450 monooxygenases (P450s) are commonly involved in biosynthesis of endogenous compounds and catabolism of xenobiotics, and their activities rely on a partner enzyme, cytochrome P450 reductase (CPR, E.C.1.6.2.4). Two CPR cDNAs, GhCPR1 and GhCPR2, were isolated from cotton (Gossypium hirsutum). They are 71% identical to each other at the amino acid sequence level and belong to the Class I and II of dicotyledonous CPRs, respectively. The recombinant enzymes reduced cytochrome c, ferricyanide and dichlorophenolindophenol (DCPIP) in an NADPH-dependent manner, and supported the activity of CYP73A25, a cinnamate 4-hydroxylase of cotton. Both GhCPR genes were widely expressed in cotton tissues, with a reduced expression level of GhCPR2 in the glandless cotton cultivar. Expression of GhCPR2, but not GhCPR1, was inducible by mechanical wounding and elicitation, indicating that the GhCPR2 is more related to defense reactions, including biosynthesis of secondary metabolites.

Cotton fibres are unicellular seed trichomes. Our previous study suggested that the cotton R2R3 MYB transcript factor GaMYB2 is a functional homologue of the Arabidopsis trichome regulator GLABRA1 (GL1). Here, the GaMYB2 promoter activity is reported in cotton (Gossypium hirsutum), tobacco (Nicotiana tabacum), and Arabidopsis plants. A 2062 bp promoter of GaMYB2 was isolated from G. arboreum, and fused to a beta-glucuronidase (GUS) reporter gene. In cotton, the GaMYB2 promoter exhibited activities in developing fibre cells and trichomes of other aerial organs, including leaves, stems and bracts. In Arabidopsis the promoter was specific to trichomes. Different from Arabidopsis and cotton that have unicellular non-glandular simple trichomes, tobacco plants contain more than one type of trichome, including multicellular simple and glandular secreting trichomes (GSTs). Interestingly, in tobacco plants the GaMYB2 promoter directed GUS expression exclusively in glandular cells of GSTs. A series of 5'-deletions revealed that a 360 bp fragment upstream to the translation initiation codon was sufficient to drive gene expression. A putative cis-element of the T/G-box was located at -233 to -214; a yeast one-hybrid assay showed that Arabidopsis bHLH protein GLABRA3 (GL3), also a trichome regulator, and GhDEL65, a GL3-like cotton protein, had high binding activities to the T/G-box motif. Overexpression of GL3 or GhDEL65 enhanced the GaMYB2 promoter activity in transgenic Arabidopsis plants. A comparison of GaMYB2 promoter specificities in trichomes of different plant species with different types of trichomes provides a tool for further dissection of plant trichome structure and development.

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.

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.