Expression and Functional Analyses of the WIP Gene Family in Arabidopsis.

The WIP family of transcription factors comprises the A1d subgroup of C2H2 zinc finger proteins. This family has six members in Arabidopsis thaliana and most of the known functions have been described by analyzing single knockout mutants. However, it has been shown that WIP2 and its closest paralogs WIP4 and WIP5 have a redundant and essential function in root meristems. It is likely that these and other WIP genes perform more, still unknown, functions. To obtain hints about these other functions, the expression of the six WIP genes was explored. Moreover, phenotypic ana-lyses of overexpressors and wip mutants revealed functions in modulating organ and cell size, stomatal density, and vasculature development.

New roles of NO TRANSMITTING TRACT and SEEDSTICK during medial domain development in Arabidopsis fruits.

The gynoecium, the female reproductive part of the flower, is key for plant sexual reproduction. During its development, inner tissues such as the septum and the transmitting tract tissue, important for pollen germination and guidance, are formed. In Arabidopsis, several transcription factors are known to be involved in the development of these tissues. One of them is NO TRANSMITTING TRACT (NTT), essential for transmitting tract formation. We found that the NTT protein can interact with several gynoecium-related transcription factors, including several MADS-box proteins, such as SEEDSTICK (STK), known to specify ovule identity. Evidence suggests that NTT and STK control enzyme and transporter-encoding genes involved in cell wall polysaccharide and lipid distribution in gynoecial medial domain cells. The results indicate that the simultaneous loss of NTT and STK activity affects polysaccharide and lipid deposition and septum fusion, and delays entry of septum cells to their normal degradation program. Furthermore, we identified KAWAK, a direct target of NTT and STK, which is required for the correct formation of fruits in Arabidopsis These findings position NTT and STK as important factors in determining reproductive competence.

Evolution of tonoplast P-ATPase transporters involved in vacuolar acidification.

Petunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H(+) P3A -ATPase, whereas PH1 is the only known eukaryoticP3B -ATPase. As there were no indications that this tonoplast pump is widespread in plants, we investigated the distribution and evolution of PH1 and PH5. We combined database mining and phylogenetic and synteny analyses of PH1- and PH5-like proteins from all kingdoms with functional analyses (mutant complementation and intracellular localization) of homologs from diverse angiosperms. We identified functional PH1 and PH5 homologs in divergent angiosperms. PH5 homologs evolved from plasma membrane P3A -ATPases, acquiring an N-terminal tonoplast-sorting sequence and new cellular function before angiosperms appeared. PH1 is widespread among seed plants and related proteins are found in some groups of bacteria and fungi and in one moss, but is absent in most algae, suggesting that its evolution involved several cases of gene loss and possibly horizontal transfer events. The distribution of PH1 and PH5 in the plant kingdom suggests that vacuolar acidification by P-ATPases appeared in gymnosperms before flowers. This implies that, next to flower color determination, vacuolar hyper-acidification is required for yet unknown processes.

TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds.

Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H(+) -ATPases in the plasma membrane, and multimeric vacuolar-type H(+) -ATPases (V-ATPases) and vacuolar H(+) -pyrophosphatases (H(+) -PPases) in endomembranes. Here, we show that reduced accumulation of proanthocyanidins (PAs) and hence the diminished brown seed coloration found in the Arabidopsis thaliana mutant transparent testa 13 (tt13) is caused by disruption of the gene encoding the P3A -ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants. Cells of the tt13 seed coat endothelium do not contain PA-filled central vacuoles as observed in the wild-type. tt13 phenocopies tt12, a mutant that is defective in vacuolar import of the PA precursor epicatechin. Our data show that vacuolar loading with PA precursors depends on TT13. Consistent with the tt13 phenotype, but in contrast to other isoforms of P-type H(+) -ATPases, TT13 localizes to the tonoplast. PA accumulation in tt13 is partially restored by expression of the tonoplast localized H(+) -PPase VHP1. Our findings indicate that the P3A -ATPase TT13 functions as a proton pump in the tonoplast of seed coat endothelium cells, and generates the driving force for TT12-mediated transport of PA precursors to the vacuole.

Update on transparent testa mutants from Arabidopsis thaliana: characterisation of new alleles from an isogenic collection.

MAIN CONCLUSION: We present a comprehensive overview on flavonoid-related phenotypes of A. thaliana tt and tds mutants, provide tools for their characterisation, increase the number of available alleles and demonstrate that tds3 is allelic to tt12 and tds5 to aha10. Flavonoid biosynthesis is one of the best-studied secondary metabolite pathways in plants. In the model system Arabidopsis thaliana it leads to the synthesis of three phenolic compound classes: flavonol glycosides, anthocyanins and proanthocyanidins (PAs). PAs appear brown in their oxidised polymeric forms, and most A. thaliana mutants impaired in flavonoid accumulation were identified through screens for lack of this seed coat pigmentation. These mutants are referred to as transparent testa (tt) or tannin-deficient seed (tds). More than 20 mutants of these types have been published, probably representing most of the genes relevant for PA accumulation in A. thaliana. However, data about the genes involved in PA deposition or oxidation are still rather scarce. Also, for some of the known mutants it is unclear if they represent additional loci or if they are allelic to known genes. For the present study, we have performed a systematic phenotypic characterisation of almost all available tt and tds mutants and built a collection of mutants in the genetic background of the accession Columbia to minimise effects arising from ecotype variation. We have identified a novel tt6 allele from a forward genetic screen and demonstrated that tds3 is allelic to tt12 and tds5 to aha10.

Leucoanthocyanidin Dioxygenase in Arabidopsis thaliana: characterization of mutant alleles and regulation by MYB-BHLH-TTG1 transcription factor complexes.

In Arabidopsis thaliana, most mutants impaired in flavonoid accumulation were identified through screens for altered seed pigmentation. Mutations in more than 20 loci have been described that can result in a transparent testa (tt) or tannin deficient seed (tds) phenotype. For some of these mutants it is still unclear if they represent additional loci or if they are allelic to known mutations. In this study, we found that tt17 is allelic to tt11 and tds4 and identified a point mutation in tt17 that affects the gene encoding Leucoanthocyanidin Dioxygenase (LDOX). The mutation results in replacement of a cysteine close to the active site of the enzyme by the hydrophobic amino acid tyrosine. Effects of this mutation on protein structure and activity are discussed in the context of LDOX sequences from various genotypes. Regulation of the LDOX promoter was analyzed and found to be directly controlled by different MYB-BHLH-TTG1 transcription factor complexes containing the BHLH factors EGL3 and TT8. Experiments with single and double loss-of-function mutants identified EGL3 and TT8 as necessary regulators of anthocyanin accumulation in developing A. thaliana seedlings.

TRANSPARENT TESTA1 interacts with R2R3-MYB factors and affects early and late steps of flavonoid biosynthesis in the endothelium of Arabidopsis thaliana seeds.

Wild type seed coats of Arabidopsis thaliana are brown due to the accumulation of proanthocyanidin pigments (PAs). The pigmentation requires activation of phenylpropanoid biosynthesis genes and mutations in some of these genes cause a yellow appearance of seeds, termed transparent testa (tt) phenotype. The TT1 gene encodes a WIP-type zinc finger protein and is expressed in the seed coat endothelium where most of the PAs accumulate in wild type plants. In this study we show that TT1 is not only required for correct expression of PA-specific genes in the seed coat, but also affects CHS, encoding the first enzyme of flavonoid biosynthesis. Many steps of this pathway are controlled by complexes of MYB and BHLH proteins with the WD40 factor TTG1. We demonstrate that TT1 can interact with the R2R3 MYB protein TT2 and that ectopic expression of TT2 can partially restore the lack in PA production in tt1. Reduced seed coat pigmentation was obtained using a TT1 variant lacking nuclear localisation signals. Based on our results we propose that the TT2/TT8/TTG1 regulon may also comprise early genes like CHS and discuss steps to further unravel the regulatory network controlling flavonoid accumulation in endothelium cells during A. thaliana seed development.

Weird fingers: functional analysis of WIP domain proteins.

WIP proteins form a plant specific subfamily of C2H2 zinc finger (ZF) proteins. In this study, we functionally characterized the WIP domain, which consists of four ZF motifs, and discuss molecular functions for WIP proteins. Mutations in each of the ZFs lead to loss of function of the TT1/WIP1 protein in Arabiopsis thaliana. SV40 type nuclear localisation signals were detected in two of the ZFs and functionally characterized using GFP fusions as well as new mutant alleles identified by TILLING. Promoter swap experiments showed that selected WIP proteins are partially able to take over TT1 function. Activity of the AtBAN promoter, a potential TT1 target, could be increased by the addition of TT1 to the TT2-TT8-TTG1 regulatory complex.

A WD40-repeat gene from Malus x domestica is a functional homologue of Arabidopsis thaliana TRANSPARENT TESTA GLABRA1.

The WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) is involved in a multitude of developmental and biochemical reactions in Arabidopsis thaliana such as the production of seed coat colour and mucilage, pigmentation by anthocyanins as well as the formation of trichomes and root hairs. In this study, a putative TTG1 homologue was isolated from apple (Malus x domestica Borkh.) showing 80.2% identity to A. thaliana TTG1 on nucleotide and 90.7% similarity on amino acid level. The MdTTG1 candidate was able to activate the AtBAN promoter in cooperation with the A. thaliana transcription factors TT2 and TT8 in A. thaliana protoplasts. This indicates that the encoded protein can be integrated into the complex that activates BAN in A. thaliana, and that a similar complex might also be present in apple. When transformed into ttg1 mutants of A. thaliana, the apple sequence was able to restore trichome growth, anthocyanin production in young seedlings as well as proanthocyanidin production in seeds. Additionally, roots of complemented mutant plants showed root hair formation resembling wild type. These results show that the studied apple WD40 gene is a functional homologue of AtTTG1 and we refer to this gene as MdTTG1.

Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase.

Flavonol synthase (FLS) (EC-number 1.14.11.23), the enzyme that catalyses the conversion of flavonols into dihydroflavonols, is part of the flavonoid biosynthesis pathway. In Arabidopsis thaliana, this activity is thought to be encoded by several loci. In addition to the FLAVONOL SYNTHASE1 (FLS1) locus that has been confirmed by enzyme activity assays, loci displaying similarity of the deduced amino acid sequences to FLS1 have been identified. We studied the putative A. thaliana FLS gene family using a combination of genetic and metabolite analysis approaches. Although several of the FLS gene family members are expressed, only FLS1 appeared to influence flavonoid biosynthesis. Seedlings of an A. thaliana fls1 null mutant (fls1-2) show enhanced anthocyanin levels, drastic reduction in flavonol glycoside content and concomitant accumulation of glycosylated forms of dihydroflavonols, the substrate of the FLS reaction. By using a leucoanthocyanidin dioxygenase (ldox) fls1-2 double mutant, we present evidence that the remaining flavonol glycosides found in the fls1-2 mutant are synthesized in planta by the FLS-like side activity of the LDOX enzyme.

Differential combinatorial interactions of cis-acting elements recognized by R2R3-MYB, BZIP, and BHLH factors control light-responsive and tissue-specific activation of phenylpropanoid biosynthesis genes.

Chalcone synthase (CHS), chalcone flavanone isomerase (CFI), flavanone 3-hydroxylase (F3H) and flavonol synthase (FLS) catalyze successive steps in the biosynthetic pathway leading to the production of flavonols. We show that in Arabidopsis thaliana all four corresponding genes are coordinately expressed in response to light, and are spatially coexpressed in siliques, flowers and leaves. Light regulatory units (LRUs) sufficient for light responsiveness were identified in all four promoters. Each unit consists of two necessary elements, namely a MYB-recognition element (MRE) and an ACGT-containing element (ACE). C1 and Sn, a R2R3-MYB and a BHLH factor, respectively, known to control tissue specific anthocyanin biosynthesis in Z. mays, were together able to activate the AtCHS promoter. This activation of the CHS promoter required an intact MRE and a newly identified sequence designated R response element (RREAtCHS) containing the BHLH factor consensus binding site CANNTG. The RRE was dispensable for light responsiveness, and the ACE was not necessary for activation by C1/Sn. These data suggest that a BHLH and a R2R3-MYB factor cooperate in directing tissue-specific production of flavonoids, while an ACE-binding factor, potentially a BZIP, and a R2R3-MYB factor work together in conferring light responsiveness.

A. thaliana TRANSPARENT TESTA 1 is involved in seed coat development and defines the WIP subfamily of plant zinc finger proteins.

Seeds of the Arabidopsis thaliana transparent testa 1 mutant (tt1) appear yellow, due to the lack of condensed tannin pigments in the seed coat. The TT1 gene was isolated by reverse genetics using an En-1 transposon mutagenized A. thaliana population. TT1 gene expression was detected in developing ovules and young seeds only, and the gene was shown to encode a nuclear protein. Mutant seeds displayed altered morphology of the seed endothelium in which brown tannin pigments accumulate in wild-type plants, indicating that TT1 is involved in the differentiation of this cell layer. When overexpressed in transgenic A. thaliana plants, TT1 caused aberrant development and organ morphology. The protein contains a novel combination of two TFIIIA-type zinc finger motifs. Closely related motifs were detected in a number of putative proteins deduced from plant genomic and EST sequences. The new protein domain containing this type of zinc finger motifs was designated WIP, according to three strictly conserved amino acid residues. Our data indicate the existence of a small gene family in A. thaliana which is defined by the occurrence of the WIP domain. WIP genes may play important roles in regulating developmental processes, including the control of endothelium differentiation.