Hanjo Hellmann

The Dual Function of Sugar Carriers: Transport and Sugar Sensing

Sucrose and its derivatives represent the major transport forms of photosynthetically assimilated carbon in plants. Sucrose synthesized in green leaves is exported via the phloem, the long-distance distribution network for assimilates, to supply nonphotosynthetic organs with energy and carbon

SUT2, a Putative Sucrose Sensor in Sieve Elements

In leaves, sucrose uptake kinetics involve high- and low-affinity components. A family of low- and high-affinity sucrose transporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-distance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping that of SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter–like proteins, named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling the yeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start codon, low expression, and lack of detectable transport activity. In contrast to LeSUT1, which is induced during the sink-to-source transition of leaves, SUT2 is more highly expressed in sink than in source leaves and is inducible by sucrose. LeSUT2 protein colocalizes with the low- and high-affinity sucrose transporters in sieve elements of tomato petioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The SUT2 gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch content and yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from them in kinetic properties, and potentially regulates the relative activity of low- and high-affinity sucrose transport into sieve elements.

AXR1-ECR1–Dependent Conjugation of RUB1 to the Arabidopsis Cullin AtCUL1 Is Required for Auxin Response

Mutations in the AXR1 gene result in a reduction in auxin response and diverse defects in auxin-regulated growth and development. In a previous study, we showed that AXR1 forms a heterodimer with the ECR1 protein. This enzyme activates the ubiquitin-related protein RUB1 in vitro. Furthermore, we showed that the Skp1-Cul1/Cdc53-F-box (SCF) subunit AtCUL1 is modified by RUB1 in vivo. In this report, we demonstrate that the formation of RUB-AtCUL1 is dependent on AXR1 and ECR1 in vivo. The expression of AXR1 and ECR1 is restricted to zones of active cell division and cell elongation, consistent with their role in growth regulation. These results provide strong support for a model in which RUB conjugation of AtCUL1 affects the function of SCF E3s that are required for auxin response.

Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function

The ubiquitin-related protein RUB/Nedd8 is conjugated to members of the cullin family of proteins in plants, animals, and fungi. In Arabidopsis, the RUB conjugation pathway consists of a heterodimeric E1 (AXR1-ECR1) and a RUB-E2 called RCE1. The cullin CUL1 is a subunit in SCF-type ubiquitin protein ligases (E3s), including the SCFTIR1 complex, which is required for response to the plant hormone auxin. Our previous studies showed that conjugation of RUB to CUL1 is required for normal SCFTIR1 function. The RING-H2 finger protein RBX1 is a subunit of SCF complexes in fungi and animals. The function of RBX1 is to bind the ubiquitin-conjugating enzyme E2 and bring it into close proximity with the E3 substrate. We have identified two Arabidopsis genes encoding RING-H2 proteins related to human RBX1. Studies of one of these proteins indicate that, as in animals and fungi, Arabidopsis RBX1 is an SCF subunit. Reduced RBX1 levels result in severe defects in growth and development. Overexpression of RBX1 increases RUB modification of CUL1. This effect is associated with reduced auxin response and severe growth defects similar to those observed in axr1 mutants. As in the axr1 mutants, RBX1 overexpression stabilizes the SCFTIR1 substrate AXR2/IAA7. The RBX1 protein is a component of SCF complexes in Arabidopsis. In addition to its direct role in SCF E3 ligase activity, RBX1 promotes the RUB modification of CUL1 and probably functions as an E3 ligase in the RUB pathway. Hypermodification of CUL1 disrupts SCFTIR1 function, suggesting that cycles of RUB conjugation and removal are important for SCF activity.

Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases in auxin regulation of embryogenesis

The AXR6 gene is required for auxin signaling in the Arabidopsis embryo and during postembryonic development. One of the effects of auxin is to stimulate degradation of the Aux/IAA auxin response proteins through the action of the ubiquitin protein ligase SCFTIR1. Here we show that AXR6 encodes the SCF subunit CUL1. The axr6 mutations affect the ability of mutant CUL1 to assemble into stable SCF complexes resulting in reduced degradation of the SCFTIR1 substrate AXR2/IAA7. In addition, we show that CUL1 is required for lateral organ initiation in the shoot apical meristem and the inflorescence meristem. These results indicate that the embryonic axr6 phenotype is related to a defect in SCF function and accumulation of Aux/IAA proteins such as BDL/IAA12. In addition, we show that CUL1 has a role in auxin response throughout the life cycle of the plant.

The RUB/Nedd8 conjugation pathway is required for early development in Arabidopsis

The related‐to‐ubiquitin (RUB) protein is post‐translationally conjugated to the cullin subunit of the SCF (SKP1, Cullin, F‐box) class of ubiquitin protein ligases. Although the precise biochemical function of RUB modification is unclear, studies indicate that the modification is important for SCF function. In Arabidopsis, RUB modification of CUL1 is required for normal function of SCFTIR1, an E3 required for response to the plant hormone auxin. In this report we show that an Arabidopsis protein called RCE1 functions as a RUB‐conjugating enzyme in vivo. A mutation in the RCE1 gene results in a phenotype like that of the axr1 mutant. Most strikingly, plants deficient in both RCE1 and AXR1 have an embryonic phenotype similar to mp and bdl mutants, previously shown to be deficient in auxin signaling. Based on these results, we suggest that the RUB‐conjugation pathway is required for auxin‐dependent pattern formation in the developing embryo. In addition, we show that RCE1 interacts directly with the RING protein RBX1 and is present in a stable complex with SCF. We propose that RBX1 functions as an E3 for RUB modification of CUL1.

Vitamin deficiencies in humans: can plant science help?

The term vitamin describes a small group of organic compounds that are absolutely required in the human diet. Although for the most part, dependency criteria are met in developed countries through balanced diets, this is not the case for the five billion people in developing countries who depend predominantly on a single staple crop for survival. Thus, providing a more balanced vitamin intake from high-quality food remains one of the grandest challenges for global human nutrition in the coming decade(s). Here, we describe the known importance of vitamins in human health and current knowledge on their metabolism in plants. Deficits in developing countries are a combined consequence of a paucity of specific vitamins in major food staple crops, losses during crop processing, and/or overreliance on a single species as a primary food source. We discuss the role that plant science can play in addressing this problem and review successful engineering of vitamin pathways. We conclude that while considerable advances have been made in understanding vitamin metabolic pathways in plants, more cross-disciplinary approaches must be adopted to provide adequate levels of all vitamins in the major staple crops to eradicate vitamin deficiencies from the global population.

Arabidopsis Has Two Redundant Cullin3 Proteins That Are Essential for Embryo Development and That Interact with RBX1 and BTB Proteins to Form Multisubunit E3 Ubiquitin Ligase Complexes in Vivo

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein–type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.

Vitamin B6: A Long Known Compound of Surprising Complexity

In recent years vitamin B6 has become a focus of research describing the compound’s critical function in cellular metabolism and stress response. For many years the sole function of vitamin B6 was considered to be that of an enzymatic cofactor. However, recently it became clear that it is also a potent antioxidant that effectively quenches reactive oxygen species and is thus of high importance for cellular well-being. In view of the recent findings, the current review takes a look back and summarizes the discovery of vitamin B6 and the elucidation of its structure and biosynthetic pathways. It provides a detailed overview on vitamin B6 both as a cofactor and a protective compound. Besides these general characteristics of the vitamin, the review also outlines the current literature on vitamin B6 derivatives and elaborates on recent findings that provide new insights into transport and catabolism of the compound and on its impact on human health.

Arabidopsis AtCUL3a and AtCUL3b Form Complexes with Members of the BTB/POZ-MATH Protein Family

The ubiquitin proteasome pathway in plants has been shown to be important for many developmental processes. The E3 ubiquitin-protein ligases facilitate transfer of the ubiquitin moiety to substrate proteins. Many E3 ligases contain cullin proteins as core subunits. Here, we show that Arabidopsis (Arabidopsis thaliana) AtCUL3 proteins interact in yeast two-hybrid and in vitro pull-down assays with proteins containing a BTB/POZ (broad complex, tramtrack, bric-a-brac/pox virus and zinc finger) motif. By changing specific amino acid residues within the proteins, critical parts of the cullin and BTB/POZ proteins are defined that are required for these kinds of interactions. In addition, we show that AtCUL3 proteins assemble with the RING-finger protein AtRBX1 and are targets for the RUB-conjugation pathway. The analysis of AtCUL3a and AtCUL3b expression as well as several BTB/POZ-MATH genes indicates that these genes are expressed in all parts of the plant. The results presented here provide strong evidence that AtCUL3a and AtCUL3b can assemble in Arabidopsis with BTB/POZ-MATH and AtRBX1 proteins to form functional E3 ligases.