The chemical compound 'Heatin' stimulates hypocotyl elongation and interferes with the Arabidopsis NIT1-subfamily of nitrilases.

Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.

ABA signalling promotes cell totipotency in the shoot apex of germinating embryos.

Somatic embryogenesis (SE) is a type of induced cell totipotency where embryos develop from vegetative tissues of the plant instead of from gamete fusion after fertilization. SE can be induced in vitro by exposing explants to growth regulators, such as the auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The plant hormone abscisic acid (ABA) has been proposed to be a downstream signalling component at the intersection between 2,4-D- and stress-induced SE, but it is not known how these pathways interact to induce cell totipotency. Here we show that 2,4-D-induced SE from the shoot apex of germinating Arabidopsis thaliana seeds is characterized by transcriptional maintenance of an ABA-dependent seed maturation pathway. Molecular-genetic analysis of Arabidopsis mutants revealed a role for ABA in promoting SE at three different levels: ABA biosynthesis, ABA receptor complex signalling, and ABA-mediated transcription, with essential roles for the ABSCISIC ACID INSENSITIVE 3 (ABI3) and ABI4 transcription factors. Our data suggest that the ability of mature Arabidopsis embryos to maintain the ABA seed maturation environment is an important first step in establishing competence for auxin-induced cell totipotency. This finding provides further support for the role of ABA in directing processes other than abiotic stress response.

Functional analyses of the CLAVATA2-like proteins and their domains that contribute to CLAVATA2 specificity.

The Arabidopsis (Arabidopsis thaliana) CLAVATA2 (CLV2) gene encodes a leucine-rich repeat receptor-like protein (RLP) that is involved in controlling the stem cell population size in the shoot apical meristem. Our previous genome-wide functional analysis of 57 AtRLP genes revealed only a few phenotypes for mutant alleles, despite screening a wide range of growth and developmental stages and assaying sensitivity to various stress responses, including susceptibility toward pathogens. To gain further insight into the biological role of AtRLPs, in particular CLV2-related AtRLP genes, we tested their ability to complement the clv2 mutant phenotype. We found that out of four close CLV2 homologs tested, AtRLP2 and AtRLP12 could functionally complement the clv2 mutant when expressed under the control of the CLV2 promoter. This indicates that the functional specificity of these three genes is determined at the level of their transcriptional regulation. Single and double mutant combinations with impaired AtRLP2 and/or AtRLP12 did not show an aberrant phenotype, suggesting that other genes are redundant with these CLV2-like genes. To understand which protein domains are essential for CLV2 function and which parts are interchangeable between related CLV2-like proteins, we performed domain-deletion and domain-swap experiments. These experiments revealed that CLV2 remains functional without the island domain, whereas the C1 and C3 regions of the leucine-rich repeat domain are essential for functionality. Analysis of domain-swap constructs showed that the C3-G region of CLV2 can be replaced by that of AtRLP38, although it could not complement the clv2 mutant under control of the CLV2 promoter. This suggests that the C3-G region is conserved among related AtRLP members, whereas the C1 domain may determine the functional specificity of CLV2.

Sequence motifs in MADS transcription factors responsible for specificity and diversification of protein-protein interaction.

Protein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana. Here, we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family, using an integrated computational and experimental approach. Plant MADS proteins have highly similar amino acid sequences, but their dimerization patterns vary substantially. Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy. Furthermore, we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species. Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions. In addition, various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity. Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs, indicating their evolutionary importance. We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization. The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and network evolution.

CLE peptide ligands and their roles in establishing meristems.

Research in the past decade revealed that peptide ligands, also called peptide hormones, play a crucial role in intercellular communication and defense response in plants. Recent studies demonstrated that a family of plant-specific genes, CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR) (CLE), which has at least 31 members in Arabidopsis genome, are able to generate extracellular peptides to regulate cell division and differentiation. A hydroxyl 12-amino acid peptide derived from the conserved CLE motif of CLV3 promotes cell differentiation, whereas another CLE-derived peptide suppresses the differentiation. These peptides probably interact with membrane-bound, leucine-rich repeat receptor-like kinases (LRR-RLKs) to execute the decision between cell proliferation and differentiation.

Combination of reversible male sterility and doubled haploid production by targeted inactivation of cytoplasmic glutamine synthetase in developing anthers and pollen.

Reversible male sterility and doubled haploid plant production are two valuable technologies in F(1)-hybrid breeding. F(1)-hybrids combine uniformity with high yield and improved agronomic traits, and provide self-acting intellectual property protection. We have developed an F(1)-hybrid seed technology based on the metabolic engineering of glutamine in developing tobacco anthers and pollen. Cytosolic glutamine synthetase (GS1) was inactivated in tobacco by introducing mutated tobacco GS genes fused to the tapetum-specific TA29 and microspore-specific NTM19 promoters. Pollen in primary transformants aborted close to the first pollen mitosis, resulting in male sterility. A non-segregating population of homozygous doubled haploid male-sterile plants was generated through microspore embryogenesis. Fertility restoration was achieved by spraying plants with glutamine, or by pollination with pollen matured in vitro in glutamine-containing medium. The combination of reversible male sterility with doubled haploid production results in an innovative environmentally friendly breeding technology. Tapetum-mediated sporophytic male sterility is of use in foliage crops, whereas microspore-specific gametophytic male sterility can be applied to any field crop. Both types of sterility preclude the release of transgenic pollen into the environment.

A plant-based chemical genomics screen for the identification of flowering inducers.

BACKGROUND: Floral timing is a carefully regulated process, in which the plant determines the optimal moment to switch from the vegetative to reproductive phase. While there are numerous genes known that control flowering time, little information is available on chemical compounds that are able to influence this process. We aimed to discover novel compounds that are able to induce flowering in the model plant Arabidopsis. For this purpose we developed a plant-based screening platform that can be used in a chemical genomics study. RESULTS: Here we describe the set-up of the screening platform and various issues and pitfalls that need to be addressed in order to perform a chemical genomics screening on Arabidopsis plantlets. We describe the choice for a molecular marker, in combination with a sensitive reporter that's active in plants and is sufficiently sensitive for detection. In this particular screen, the firefly Luciferase marker was used, fused to the regulatory sequences of the floral meristem identity gene APETALA1 (AP1), which is an early marker for flowering. Using this screening platform almost 9000 compounds were screened, in triplicate, in 96-well plates at a concentration of 25 µM. One of the identified potential flowering inducing compounds was studied in more detail and named Flowering1 (F1). F1 turned out to be an analogue of the plant hormone Salicylic acid (SA) and appeared to be more potent than SA in the induction of flowering. The effect could be confirmed by watering Arabidopsis plants with SA or F1, in which F1 gave a significant reduction in time to flowering in comparison to SA treatment or the control. CONCLUSIONS: In this study a chemical genomics screening platform was developed to discover compounds that can induce flowering in Arabidopsis. This platform was used successfully, to identify a compound that can speed-up flowering in Arabidopsis.

Cross-Talk Between Sporophyte and Gametophyte Generations Is Promoted by CHD3 Chromatin Remodelers in Arabidopsis thaliana.

Angiosperm reproduction requires the integrated development of multiple tissues with different genotypes. To achieve successful fertilization, the haploid female gametophytes and diploid ovary must coordinate their development, after which the male gametes must navigate through the maternal sporophytic tissues to reach the female gametes. After fertilization, seed development requires coordinated development of the maternal diploid integuments, the triploid endosperm, and the diploid zygote. Transcription and signaling factors contribute to communication between these tissues, and roles for epigenetic regulation have been described for some of these processes. Here we identify a broad role for CHD3 chromatin remodelers in Arabidopsis thaliana reproductive development. Plants lacking the CHD3 remodeler, PICKLE, exhibit various reproductive defects including abnormal development of the integuments, female gametophyte, and pollen tube, as well as delayed progression of ovule and embryo development. Genetic analyses demonstrate that these phenotypes result from loss of PICKLE in the maternal sporophyte. The paralogous gene PICKLE RELATED 2 is preferentially expressed in the endosperm and acts antagonistically with respect to PICKLE in the seed: loss of PICKLE RELATED 2 suppresses the large seed phenotype of pickle seeds. Surprisingly, the alteration of seed size in pickle plants is sufficient to determine the expression of embryonic traits in the seedling primary root. These findings establish an important role for CHD3 remodelers in plant reproduction and highlight how the epigenetic status of one tissue can impact the development of genetically distinct tissues.

Mis-expression of the CLV3/ESR-like gene CLE19 in Arabidopsis leads to a consumption of root meristem.

Mild heat shock treatment (32 degrees C) of isolated Brassica napus microspores triggers a developmental switch from pollen maturation to embryo formation. This in vitro system was used to identify genes expressed in globular to heart-shape transition embryos. One of the genes isolated encodes a putative extra-cellular protein that exhibits high sequence similarity with the in silico identified CLV3/ESR-related 19 polypeptide from Arabidopsis (AtCLE19) and was therefore named BnCLE19. BnCLE19 is expressed in the primordia of cotyledons, sepals and cauline leaves, and in some pericycle cells in the root maturation zone. Mis-expression of BnCLE19 or AtCLE19 in Arabidopsis under the control of the CaMV 35S promoter resulted in a dramatic consumption of the root meristem, the formations of pin-shaped pistils and vascular islands. These results imply a role of CLE19 in promoting cell differentiation or inhibiting cell division.

New aspects of CLAVATA2, a versatile gene in the regulation of Arabidopsis development.

In order to unravel the functions of receptor-like proteins in Arabidopsis (AtRLPs), including the CLAVATA2 (CLV2) receptor, we employed a reverse genetics approach. In previous studies, we assembled a genome-wide collection of AtRLP gene T-DNA insertion mutants and reported on the biological roles of a few AtRLPs closely related to CLV2. In this study, we showed that over-expression of CLV2 in wild-type plants unexpectedly resulted in a multi-carpel phenotype, mimicking the clv2 mutant, which suggests a co-suppression effect on the endogenous CLV2. We also found evidence that the phenotypes of various clv2 alleles are significantly affected by the genetic background of Arabidopsis ecotypes, which, however, did not depend on the ER locus.

CLE peptide signaling during plant development.

Peptide signaling in plants is a rapid developing area of research which focuses on so called peptide hormones. These signaling molecules are utilized for inter-cellular communication in different developmental processes, beside the usage of the more well-known phytohormones. Probably the best studied peptide ligands in plants are the CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) proteins. This family of signaling polypeptides is comprised of 32 members in Arabidopsis and, with the exception of the presence of related proteins in some parasitic worms, is restricted to the plant kingdom. CLV3 is one of the founding CLE genes and is involved in stem cell niche maintenance in apical meristems during plant development. While the CLV signaling pathway is well characterized with the identification of three receptors and a stem-cell-promoting transcription factor as target, the functioning of other family members is not or poorly understood. The recent discoveries of a new type of receptor involved in CLV signaling and a functional pathway for CLE40 in root development mark the rapid progress that is made in the area of CLE peptide signaling. This review gives an overview how CLE peptides are used as signaling molecules, and how they are involved in cell-to-cell communication in concert with different known and unknown receptors in a range of developmental processes during plant development.

Receptor-like proteins: searching for functions.

Receptor-like proteins (RLPs) are cell surface receptors, which are composed of several distinct domains including a signal peptide, an extracellular leucine-rich repeat (LRR) region, a transmembrane domain and a short cytoplasmic tail. RLPs are implicated in plant growth and development as well as in disease resistance. Our previous genome-wide functional analysis of 57 Arabidopsis RLP genes (AtRLPs) revealed that mutant phenotypes were only observed for a few genes including the two reported genes CLAVATA2 (CLV2) and TOO MANY MOUTHS, despite the wide range of growth and developmental stages and treatments that were tested. In a recent study, we reported on further insights into the biological role of a few AtRLPs closely-related to CLV2. Two AtRLPs (AtRLP2 and AtRLP12), which share high sequence similarity with CLV2, were found to be able to rescue the clv2 mutant phenotype when expressed under the control of the CLV2 promoter, suggesting that the specialization among CLV2, AtRLP2 and AtRLP12 is largely ascribed to differences in their expression patterns. Our data further indicated that the island domain of CLV2 is dispensable for its function and the C3-F domain of CLV2 could be replaced by that of AtRLP38. In this Addendum, we are elaborating on further strategies concerning the function of largely unknown AtRLPs.

The CLAVATA3/ESR motif of CLAVATA3 is functionally independent from the nonconserved flanking sequences.

It is believed that CLAVATA3 (CLV3) encodes a peptide ligand that interacts with the CLV1/CLV2 receptor complex to limit the number of stem cells in the shoot apical meristem of Arabidopsis thaliana; however, the exact composition of the functional CLV3 product remains a mystery. A recent study on CLV3 shows that the CLV3/ESR (CLE) motif, together with the adjacent C-terminal sequence, is sufficient to execute CLV3 function when fused behind an N-terminal sequence of ERECTA. Here we show that most of the sequences flanking the CLE motif of CLV3 can be deleted without affecting CLV3 function. Using a liquid culture assay, we demonstrate that CLV3p, a synthetic peptide corresponding to the CLE motif of CLV3, is able to restrict the size of the shoot apical meristem in clv3 seedlings but not in clv1 seedlings. In accordance with this decrease in meristem size, application of CLV3p to in vitro-grown clv3 seedlings restricts the expression of the stem cell-promoting transcription factor WUSCHEL. Thus, we propose that the CLE motif is the functional region of CLV3 and that this region acts independently of its adjacent sequences.

The 14-amino acid CLV3, CLE19, and CLE40 peptides trigger consumption of the root meristem in Arabidopsis through a CLAVATA2-dependent pathway.

CLAVATA3 (CLV3), CLV3/ESR19 (CLE19), and CLE40 belong to a family of 26 genes in Arabidopsis thaliana that encode putative peptide ligands with unknown identity. It has been shown previously that ectopic expression of any of these three genes leads to a consumption of the root meristem. Here, we show that in vitro application of synthetic 14-amino acid peptides, CLV3p, CLE19p, and CLE40p, corresponding to the conserved CLE motif, mimics the overexpression phenotype. The same result was observed when CLE19 protein was applied externally. Interestingly, clv2 failed to respond to the peptide treatment, suggesting that CLV2 is involved in the CLE peptide signaling. Crossing of the CLE19 overexpression line with clv mutants confirms the involvement of CLV2. Analyses using tissue-specific marker lines revealed that the peptide treatments led to a premature differentiation of the ground tissue daughter cells and misspecification of cell identity in the pericycle and endodermis layers. We propose that these 14-amino acid peptides represent the major active domain of the corresponding CLE proteins, which interact with or saturate an unknown cell identity-maintaining CLV2 receptor complex in roots, leading to consumption of the root meristem.