Search

SUMMARY: Plants supply both food and medicinal compounds, which are ascribed to diverse metabolites produced by plants. However, studies on domestication‐driven changes in the metabolome and genetic basis of bioactive molecules in perennial fruit trees are generally lacking. Here, we conducted multidimensional analyses revealing a singular domestication event involving the genomic and metabolomic selection of jujube trees (Ziziphus jujuba Mill.). The genomic selection for domesticated genes was highly enriched in metabolic pathways, including carbohydrates and specialized metabolism. Domesticated metabolome profiling indicated that 187 metabolites exhibited significant divergence as a result of directional selection. Malic acid was directly selected during domestication, and the simultaneous selection of specialized metabolites, including triterpenes, consequently lead to edible properties. Cyclopeptide alkaloids (CPAs) were specifically targeted for the divergence between dry and fresh cultivars. We identified 1080 significantly associated loci for 986 metabolites. Among them, 15 triterpenes were directly selected at six major loci, allowing the identification of a homologous cluster containing seven 2,3‐oxidosqualene cyclases (OSCs). An OSC gene was found to contribute to the reduction in the content of triterpenes during domestication. The complete pathway for synthesizing ursolic acid was dissected by integration of the metabolome and transcriptome. Additionally, an N‐methyltransferase involved in the biosynthesis of CPA and responsible for inter‐cultivar content variation was identified. The present study promotes our understanding of the selection process of the global metabolome subsequent to fruit tree domestication and facilitates the genetic manipulation of specialized metabolites to enhance their edible traits. Significance Statement: The present study promotes our understanding of the selection process of the global metabolome subsequent to fruit tree domestication and facilitates the genetic manipulation of specialized metabolites to enhance their edible traits. [ABSTRACT FROM AUTHOR]

SUMMARY: The acidification of plant vacuoles is of great importance for various physiological processes, as a multitude of secondary active transporters utilize the proton gradient established across the vacuolar membrane. Vacuolar‐type H+‐translocating ATPases and a pyrophosphatase are thought to enable vacuoles to accumulate protons against their electrochemical potential. However, recent studies pointed to the ATPase located at the trans‐Golgi network/early endosome (TGN/EE) to contribute to vacuolar acidification in a manner not understood as of now. Here, we combined experimental data and computational modeling to test different hypotheses for vacuolar acidification mechanisms. For this, we analyzed different models with respect to their ability to describe existing experimental data. To better differentiate between alternative acidification mechanisms, new experimental data have been generated. By fitting the models to the experimental data, we were able to prioritize the hypothesis in which vesicular trafficking of Ca2+/H+‐antiporters from the TGN/EE to the vacuolar membrane and the activity of ATP‐dependent Ca2+‐pumps at the tonoplast might explain the residual acidification observed in Arabidopsis mutants defective in vacuolar proton pump activity. The presented modeling approach provides an integrative perspective on vacuolar pH regulation in Arabidopsis and holds potential to guide further experimental work. Significance Statement: The study represents the first integrative computational model of pH homeostasis in the vacuole of Arabidopsis thaliana. With its aid and based on experimental data, we were able to study the feasibility of different hypotheses for experimental observations of pH changes after perturbation. [ABSTRACT FROM AUTHOR]

Summary: The accumulation of secondary metabolites and the regulation of tissue acidity contribute to the important traits of grape berry and influence plant performance in response to abiotic and biotic factors. In several plant species a highly conserved MYB–bHLH–WD (MBW) transcriptional regulatory complex controls flavonoid pigment synthesis and transport, and vacuolar acidification in epidermal cells. An additional component, represented by a WRKY‐type transcription factor, physically interacts with the complex increasing the expression of some target genes and adding specificity for other targets. Here we investigated the function of MBW(W) complexes involving two MYBs (VvMYB5a and VvMYB5b) and the WRKY factor VvWRKY26 in grapevine (Vitis vinifera L.). Using transgenic grapevine plants we showed that these complexes affected different aspects of morphology, plant development, pH regulation, and pigment accumulation. Transcriptomic analysis identified a core set of putative target genes controlled by VvMYB5a, VvMYB5b, and VvWRKY26 in different tissues. Our data indicated that VvWRKY26 enhances the expression of selected target genes induced by VvMYB5a/b. Among these targets are genes involved in vacuolar hyper‐acidification, such as the P‐type ATPases VvPH5 and VvPH1, and trafficking, and genes involved in the biosynthesis of flavonoids. In addition, VvWRKY26 is recruited specifically by VvMYB5a, reflecting the functional diversification of VvMYB5a and VvMYB5b. The expression of MBWW complexes in vegetative organs, such as leaves, indicates a possible function of vacuolar hyper‐acidification in the repulsion of herbivores and/or in developmental processes, as shown by defects in transgenic grape plants where the complex is inactivated. Significance Statement: The two R2R3‐MYB paralogs VvMYB5a and VvMYB5b and the WRKY‐type transcription factor VvWRKY26 are involved in the regulation of vacuolar hyper‐acidification during early development of grapevine organs and the alteration of their expression affects morphology, plant development, and pigment accumulation. VvWRKY26 is part of the MYB5‐driven MBWW complexes, enhances the expression of VvMYB5a and VvMYB5b target genes, and is apparently recruited specifically by VvMYB5a, therefore contributing to the functional diversification of VvMYB5a and VvMYB5b. [ABSTRACT FROM AUTHOR]

We have identified in apple ( Malus × domestica) three chalcone synthase ( CHS) genes. In order to understand the functional redundancy of this gene family RNA interference knockout lines were generated where all three of these genes were down-regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down-regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS-silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS-silenced line was smaller than the 'Royal Gala' controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased rates of auxin transport in a CHS-silenced line compared with the 'Royal Gala' control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development. [ABSTRACT FROM AUTHOR]

We present an investigation of anthocyanin regulation over the entire petunia plant, determining the mechanisms governing complex floral pigmentation patterning and environmentally induced vegetative anthocyanin synthesis. DEEP PURPLE ( DPL) and PURPLE HAZE ( PHZ) encode members of the R2R3-MYB transcription factor family that regulate anthocyanin synthesis in petunia, and control anthocyanin production in vegetative tissues and contribute to floral pigmentation. In addition to these two MYB factors, the basic helix-loop-helix (bHLH) factor ANTHOCYANIN1 (AN1) and WD-repeat protein AN11, are also essential for vegetative pigmentation. The induction of anthocyanins in vegetative tissues by high light was tightly correlated to the induction of transcripts for PHZ and AN1. Interestingly, transcripts for PhMYB27, a putative R2R3-MYB active repressor, were highly expressed during non-inductive shade conditions and repressed during high light. The competitive inhibitor PhMYBx (R3-MYB) was expressed under high light, which may provide feedback repression. In floral tissues DPL regulates vein-associated anthocyanin pigmentation in the flower tube, while PHZ determines light-induced anthocyanin accumulation on exposed petal surfaces (bud-blush). A model is presented suggesting how complex floral and vegetative pigmentation patterns are derived in petunia in terms of MYB, bHLH and WDR co-regulators. [ABSTRACT FROM AUTHOR]

Here we demonstrate that GmMYB176 regulates CHS8 expression and affects isoflavonoid synthesis in soybean. We previously established that CHS8 expression determines the isoflavonoid level in soybean seeds by comparing the transcript profiles of cultivars with different isoflavonoid contents. In the present study, a functional genomic approach was used to identify the factor that regulates CHS8 expression and isoflavonoid synthesis. Candidate genes were cloned, and co-transfection assays were performed in Arabidopsis leaf protoplasts. The results showed that GmMYB176 can trans-activate the CHS8 promoter with maximum activity. Transient expression of GmMYB176 in soybean embryo protoplasts increased endogenous CHS8 transcript levels up to 169-fold after 48 h. GmMYB176 encodes an R1 MYB protein, and is expressed in soybean seed during maturation. Furthermore, GmMYB176 recognizes a 23 bp motif containing a TAGT(T/A)(A/T) sequence within the CHS8 promoter. A subcellular localization study confirmed nuclear localization of GmMYB176. A predicted pST binding site for 14-3-3 protein is required for subcellular localization of GmMYB176. RNAi silencing of GmMYB176 in hairy roots resulted in reduced levels of isoflavonoids, showing that GmMYB176 is necessary for isoflavonoid biosynthesis. However, over-expression of GmMYB176 was not sufficient to increase CHS8 transcript and isoflavonoid levels in hairy roots. We conclude that an R1 MYB transcription factor, GmMYB176, regulates CHS8 expression and isoflavonoid synthesis in soybean. [ABSTRACT FROM AUTHOR]

In maize, human selection for yellow endosperm has led to diversification of grain carotenoid content and composition. This variation has remained largely untapped in modern breeding programs that have focused nearly exclusively on yield gains. In this paper, we show that carotenoid accumulation patterns differ in maize embryo and endosperm tissues, and that this tissue-specific accumulation is largely mediated through differential expression of genes encoding lycopene β-cyclase and lycopene ε-cyclase ( LcyB and LcyE). In the absence of LCYB function, LCYE produces a number of unusual carotenes, including δ-carotene, ε-carotene and lactucaxanthin (ε,ε-carotene-3,3′-diol), in endosperm tissue. A similar carotene cyclization profile is seen when LcyE is introduced into lycopene-accumulating Escherichia coli cells, suggesting that the carotenoid profile in the endosperm tissue of the lcyB mutant is largely due to the activity of LCYE in the absence of LCYB. Using site-directed mutagenesis of LcyE, critical amino acids were defined that regulate the product specificity of the enzyme. Finally, we show that several genes encoding enzymes in isoprenoid and carotenoid biosynthesis are probably subject to negative transcriptional regulation, mediated by a carotenoid or a molecule derived from a carotenoid. The implications of these findings with respect to breeding for carotenoid composition in maize grain are discussed. [ABSTRACT FROM AUTHOR]

Numerous GAST-like genes have been identified in various plant species. All code for small proteins with a conserved C-terminal region in which 12 cysteines are located in exactly the same positions. We have previously identified five gibberellin (GA)-induced GAST1-like genes in petunia, GIP1– 5. GIP2 is expressed in elongating zones, and its suppression in transgenic petunia plants inhibits stem elongation, suggesting a role for the protein in GA-induced cell growth. However, nothing is known about the biochemical activity of GIP2 or any other GAST-like protein. As all contain putative catalytic disulfide bonds (putative redox-active cysteines), we speculated that they might be involved in redox regulation. Expression analysis of GIP2, GIP4 and GIP5 revealed that they are induced by H2O2. To study whether GIP2 modulates H2O2 levels, we generated transgenic petunia plants expressing GIP2 under the regulation of the ubiquitous CaMV 35S promoter. The transgene reduced H2O2 levels in leaves following wounding. It also reduced the levels of H2O2 in guard cells following osmotic stress and ABA treatments, leading to the suppression of stomatal closure. In addition, the transgene promoted stem and corolla elongation. As reactive oxygen species (ROS) are involved in cell elongation, we suggest that GIP2 affects growth by regulating the levels of ROS. As all known GAST-like proteins contain putative redox-active cysteines, they may all act as antioxidants. [ABSTRACT FROM AUTHOR]

MADS-domain transcription factors are essential for proper flower and seed development in angiosperms and their role in determination of floral organ identity can be described by the ‘ABC model’ of flower development. Recently, close relatives of the B-type genes were identified by phylogenetic studies, which are referred to as Bsister (Bs) genes. Here, we report the isolation and characterization of a MADS-box Bs member from petunia, designated FBP24. An fbp24 knock-down line appeared to closely resemble the Arabidopsis Bs mutant abs and a detailed and comparative analysis led to the conclusion that both FBP24 and ABS are necessary to determine the identity of the endothelial layer within the ovule. Protein interaction studies revealed the formation of higher-order complexes between Bs–C–E and Bs–D–E type MADS-box proteins, suggesting involvement of these specific complexes in determination of endothelium identity. However, although there are many similarities between the two genes and their products and functions, interestingly FBP24 cannot replace ABS in Arabidopsis. The results presented here demonstrate the importance of the comparative analysis of key regulatory genes in various model systems to fully understand all aspects of plant development. [ABSTRACT FROM AUTHOR]

Decreased arsenate [As(V)] uptake is the major mechanism of naturally selected As(V) hypertolerance in plants. However, As(V)-hypertolerant ecotypes also show enhanced rates of phytochelatin (PC) accumulation, suggesting that improved sequestration might additionally contribute to the hypertolerance phenotype. Here, we show that enhanced PC-based sequestration in As(V)-hypertolerant Holcus lanatus is not due to an enhanced capacity for PC synthesis as such, but to increased As(V) reductase activity. Vacuolar transport of arsenite -thiol complexes was equal in both ecotypes. Based on homology with the yeast As(V) reductase, Acr2p, we identified a Cdc25-like plant candidate, HlAsr , and confirmed the As(V) reductase activity of both HlAsr and the homologous protein from Arabidopsis thaliana. The gene appeared to be As(V)-inducible and its expression was enhanced in the As(V)-hypertolerant H. lanatus ecotype, compared with the non-tolerant ecotype. Homologous ectopic overexpression of the At ASR cDNA in A. thaliana produced a dual phenotype. It improved tolerance to mildly toxic levels of As(V) exposure, but caused hypersensitivity to more toxic levels. Arabidopsis asr T-DNA mutants showed increased As(V) sensitivity at low exposure levels and enhanced arsenic retention in the root. It is argued that, next to decreased uptake, enhanced expression of Hl ASR might act as an additional determinant of As(V) hypertolerance and As transport in H. lanatus. [ABSTRACT FROM AUTHOR]

The pl1 gene encodes a MYB-related transcriptional activator committed to the regulation of anthocyanin biosynthesis in maize. Here, we report the genetic and molecular characterisation of pl-bol3, an Andean allele displaying features that make it different from all the known pl1 alleles. pl-bol3 has partial, light-independent expression, and it is active mainly in the juvenile phase of growth. It has a complex molecular structure, containing multiple pl1 gene copies, thus being the first complex locus discovered in the c1/pl1 family. Although the composite genes of the complex locus encode proteins identical to other functional PL1 proteins, the putative promoters of the pl-bol3 gene are different from the promoters of Pl-Rhoades ( Pl-Rh) and pl1 sun-red alleles. The intensity and the tissue specificity of anthocyanin production directed by pl-bol3 differ significantly from that of Pl-Rh and the original pl-W22, and are specified by the interaction of pl-bol3 with the different r1/b1 gene family members and the competence of pl-bol3 to different pigment tissues. This allele represents a natural example of gene duplication and diversification of expression, giving rise to a significant change in phenotype and, in this way, is analogous to the complex r1 locus in maize. Analysis of the pl-bol3 allele contributes to understanding the generation of diversity associated with multiple-copy genes and the molecular basis of allele-specific gene expression. [ABSTRACT FROM AUTHOR]

Summary Cytokinins (CKs) are phytohormones that play an important role in plant growth and development. Although the first naturally produced CK, zeatin, was isolated almost four decades ago, no endogenous gene has been shown to produce active CKs in planta . In an activation tagging experiment we have identified a petunia line that showed CK-specific effects including enhanced shooting, reduced apical dominance and delayed senescence and flowering. This phenotype correlated with the enhanced expression of a gene we labelled Sho (Shooting ). Sho , which encodes a protein with homology to isopentenyl transferases (IPTs), also causes CK-specific effects when expressed in other plant species. In contrast to the ipt gene from Agrobacterium , which primarily increases zeatin levels, Sho expression in petunia and tobacco especially enhances the levels of certain N6 -(Δ2 -isopentenyl) adenosine (2iP) derivatives. Our data suggest that Sho encodes a plant enzyme whose activity is sufficient to produce active CKs in plants. [ABSTRACT FROM AUTHOR]

SummarySPY acts as a negative regulator of gibberellin (GA) action in Arabidopsis, but its mode of action and regulation are still unknown. SPY over-expression in transgenic petunia plants affected various GA-regulated processes, including seed germination, shoot elongation, flower initiation, flower development and the expression of a GA-induced gene, GIP. A similar phenotype was obtained when wild-type petunia plants were treated with the GA-biosynthesis inhibitor, paclobutrazol. The N-terminus of SPY contains tetratricopeptide repeats (TPR). TPR motifs participate in protein–protein interactions, suggesting that SPY is part of a multiprotein complex. To test this hypothesis, we over-expressed the SPY's TPR region without the catalytic domain in transgenic petunia and generated a dominant-negative SPY mutant. The transgenic seeds were able to germinate on paclobutrazol, suggesting an enhanced GA signal. We cloned the petunia SPY homologue, PhSPY, and showed that its mRNA level is not affected by GA or ABA. The results of this study support the role of SPY as a negative regulator of GA action, suggest that the TPR domain is required for the interaction with other proteins to form an active complex and indicate that different plants use similar mechanisms to transduce the GA signal. [ABSTRACT FROM AUTHOR]

Summary The introduction of chalcone synthase A transgenes into petunia plants can result in degradation of chalcone synthase A RNAs and loss of chalcone synthase, a process called cosuppression or post-transcriptional gene silencing. Here we show that the RNA degradation is associated with changes in premRNA processing, i.e. loss of tissue specificity in transcript cleavage patterns, accumulation of unspliced molecules, and use of template-specific secondary poly(A) sites. These changes can also be observed at a lower level in leaves but not flowers of nontransgenic petunias. Based on this, a model is presented of how transgenes may disturb the carefully evolved, developmentally controlled post-transcriptional regulation of chalcone synthase gene expression by influencing the survival rate of the endogenous and their own mRNA. [ABSTRACT FROM AUTHOR]

Examines the distinct features of post-transcriptional gene silencing by antisense transgenes. Application of antisense transgenes in plants; Inhibition of gene expression; Correlation between RNA levels and silencing degree.

The natural bicolor floral traits of the horticultural petunia ( Petunia hybrida) cultivars Picotee and Star are caused by the spatial repression of the chalcone synthase A ( CHS-A) gene, which encodes an anthocyanin biosynthetic enzyme. Here we show that Picotee and Star petunias carry the same short interfering RNA (siRNA)-producing locus, consisting of two intact CHS-A copies, PhCHS-A1 and PhCHS-A2, in a tandem head-to-tail orientation. The precursor CHS mRNAs are transcribed from the two CHS-A copies throughout the bicolored petals, but the mature CHS mRNAs are not found in the white tissues. An analysis of small RNAs revealed the accumulation of siRNAs of 21 nucleotides that originated from the exon 2 region of both CHS-A copies. This accumulation is closely correlated with the disappearance of the CHS mRNAs, indicating that the bicolor floral phenotype is caused by the spatially regulated post-transcriptional silencing of both CHS-A genes. Linkage between the tandemly arranged CHS-A allele and the bicolor floral trait indicates that the CHS-A allele is a necessary factor to confer the trait. We suppose that the spatially regulated production of siRNAs in Picotee and Star flowers is triggered by another putative regulatory locus, and that the silencing mechanism in this case may be different from other known mechanisms of post-transcriptional gene silencing in plants. A sequence analysis of wild Petunia species indicated that these tandem CHS-A genes originated from Petunia integrifolia and/or Petunia inflata, the parental species of P. hybrida, as a result of a chromosomal rearrangement rather than a gene duplication event. [ABSTRACT FROM AUTHOR]