Burkhard Schulz

Plant ABC proteins – a unified nomenclature and updated inventory

The ABC superfamily comprises both membrane-bound transporters and soluble proteins involved in a broad range of processes, many of which are of considerable agricultural, biotechnological and medical potential. Completion of the Arabidopsis and rice genome sequences has revealed a particularly large and diverse complement of plant ABC proteins in comparison with other organisms. Forward and reverse genetics, together with heterologous expression, have uncovered many novel roles for plant ABC proteins, but this progress has been accompanied by a confusing proliferation of names for plant ABC genes and their products. A consolidated nomenclature will provide much-needed clarity and a framework for future research.

The Arabidopsis thaliana ABC transporter AtMRP5 controls root development and stomata movement

In the present study, we investigated a new member of the ABC transporter superfamily of Arabidopsis thaliana, AtMRP5. AtMRP5 encodes a 167 kDa protein and exhibits low glutathione conjugate and glucuronide conjugate transport activity. Promotor‐β‐glucuronidase fusion constructs showed that AtMRP5 is expressed mainly in the vascular bundle and in the epidermis, especially guard cells. Using reverse genetics, we identified a plant with a T‐DNA insertion in AtMRP5 (mrp5‐1). mrp5‐1 exhibited decreased root growth and increased lateral root formation. Auxin levels in the roots of mrp5‐1 plants were increased. This observation may indicate that AtMRP5 works as an auxin conjugate transporter or that mutant plants are affected in ion uptake, which may lead to changes in auxin concentrations. Experiments on epidermal strips showed that in contrast to wild type, the sulfonylurea glibenclamide had no effect on stomatal opening in mrp5‐1 plants. This result strongly suggests that AtMRP5 may also function as an ion channel regulator.

Immunophilin-like TWISTED DWARF1 Modulates Auxin Efflux Activities of Arabidopsis P-glycoproteins

The immunophilin-like protein TWISTED DWARF1 (TWD1/FKBP42) has been shown to physically interact with the multidrug resistance/P-glycoprotein (PGP) ATP-binding cassette transporters PGP1 and PGP19 (MDR1). Overlapping phenotypes of pgp1/pgp19 and twd1 mutant plants suggested a positive regulatory role of TWD1 in PGP-mediated export of the plant hormone auxin, which controls plant development. Here, we provide evidence at the cellular and plant levels that TWD1 controls PGP-mediated auxin transport. twd1 and pgp1/pgp19 cells showed greatly reduced export of the native auxin indole-3-acetic acid (IAA). Constitutive overexpression of PGP1 and PGP19, but not TWD1, enhanced auxin export. Coexpression of TWD1 and PGP1 in yeast and mammalian cells verified the specificity of the regulatory effect. Employing an IAA-specific microelectrode demonstrated that IAA influx in the root elongation zone was perturbed and apically shifted in pgp1/pgp19 and twd1 roots. Mature roots of pgp1/pgp19 and twd1 plants revealed elevated levels of free IAA, which seemed to account for agravitropic root behavior. Our data suggest a novel mode of PGP regulation via FK506-binding protein-like immunophilins, implicating possible alternative strategies to overcome multidrug resistance.

The Arabidopsis Plastidic Glucose 6-Phosphate/Phosphate Translocator GPT1 Is Essential for Pollen Maturation and Embryo Sac Development

Plastids of nongreen tissues can import carbon in the form of glucose 6-phosphate via the glucose 6-phosphate/phosphate translocator (GPT). The Arabidopsis thaliana genome contains two homologous GPT genes, AtGPT1 and AtGPT2. Both proteins show glucose 6-phosphate translocator activity after reconstitution in liposomes, and each of them can rescue the low-starch leaf phenotype of the pgi1 mutant (which lacks plastid phosphoglucoisomerase), indicating that the two proteins are also functional in planta. AtGPT1 transcripts are ubiquitously expressed during plant development, with highest expression in stamens, whereas AtGPT2 expression is restricted to a few tissues, including senescing leaves. Disruption of GPT2 has no obvious effect on growth and development under greenhouse conditions, whereas the mutations gpt1-1 and gpt1-2 are lethal. In both gpt1 lines, distorted segregation ratios, reduced efficiency of transmission in males and females, and inability to complete pollen and ovule development were observed, indicating profound defects in gametogenesis. Embryo sac development is arrested in the gpt1 mutants at a stage before the fusion of the polar nuclei. Mutant pollen development is associated with reduced formation of lipid bodies and small vesicles and the disappearance of dispersed vacuoles, which results in disintegration of the pollen structure. Taken together, our results indicate that GPT1-mediated import of glucose 6-phosphate into nongreen plastids is crucial for gametophyte development. We suggest that loss of GPT1 function results in disruption of the oxidative pentose phosphate cycle, which in turn affects fatty acid biosynthesis.

Compensation of decreased triose phosphate/phosphate translocator activity by accelerated starch turnover and glucose transport in transgenic tobacco

Abstract. Tobacco (Nicotiana tabacum L.) plants were transformed with an antisense construct of the chloroplast triose phosphate/phosphate translocator (TPT). Three transformant lines of the T4 progeny, which showed a large decrease in the transcript level of the TPT were used for further biochemical and physiological characterisation. In all antisense lines tested, TPT transport activity was diminished by 50–70% compared with the wild type (WT). Despite this high reduction in the transport capacity, αTPT plants lacked any visible phenotype. Hexokinase and α-amylase activities were increased in αTPT plants compared with the WT, whereas activities of ribulose-1,5-bisphosphate carboxylase/oxygenase and ADP-glucose pyrophosphorylase (AGPase) were not affected. At the end of a 14-h light period, leaf starch contents in αTPT lines were similar to those of the WT and controls, indicating that a decrease in the TPT had no effect on starch accumulation. Sucrose contents were diminished by more than 50% in αTPT lines compared with control plants. The time course of starch accumulation revealed a transient increase in the starch content in a selected αTPT line after 6 h in the light, followed by a decrease towards the end of the light period. Labelling with 14C indicated that during the dark and light (late afternoon) periods starch is mobilised at higher rates in αTPT lines than in the controls. Glucose/fructose ratios at the end of the dark period were increased from 1.2 in control plants to 2 in αTPT lines indicating increased amylolytic starch degradation. Initial rates of [14C] glucose transport in isolated chloroplasts were increased by a factor of 2–3 in αTPT plants compared with the WT. Rates of CO2 assimilation were substantially diminished in the αTPT lines in high CO2 and low O2, but remained unaffected in ambient CO2. The rate of photosynthetic electron transport during the induction of photosynthesis in saturating CO2 exhibited pronounced oscillations only in WT and control plants. Oscillations were less pronounced in αTPT plants, indicating that phosphate limitation of photosynthesis is lowered in αTPT plants compared with the WT. It is proposed that photoassimilates are more readily directed into starch biosynthesis in αTPT plants. This is supported by determinations of 3-phosphoglycerate levels (an activator of AGPase) during the transition from dark to light in high CO2.

Brassinosteroid control of sex determination in maize

Brassinosteroids (BRs) are plant hormones that regulate growth and development. They share structural similarities with animal steroids, which are decisive factors of sex determination. BRs are known to regulate morphogenesis and environmental stress responses, but their involvement in sex determination in plants has been only speculative. We show that BRs control sex determination in maize revealed through characterization of the classical dwarf mutant nana plant1 (na1), which also feminizes male flowers. na1 plants carry a loss-of-function mutation in a DET2 homolog—a gene in the BR biosynthetic pathway. The mutant accumulates the DET2-specific substrate (24R)-24-methylcholest-4-en-3-one with a concomitant decrease of downstream BR metabolites. Treatment of wild-type maize plants with BR biosynthesis inhibitors completely mimicked both dwarf and tasselseed phenotypes of na1 mutants. Tissue-specific na1 expression in anthers throughout their development supports the hypothesis that BRs promote masculinity of the male inflorescence. These findings suggest that, in the monoecious plant maize, BRs have been coopted to perform a sex determination function not found in plants with bisexual flowers.

Family business: the multidrug-resistance related protein (MRP) ABC transporter genes in Arabidopsis thaliana

Abstract. Despite the completion of the sequencing of the entire genome of Arabidopsis thaliana (L.) Heynh., the exact determination of each single gene and its function remains an open question. This is especially true for multigene families. An approach that combines analysis of genomic structure, expression data and functional genomics to ascertain the role of the members of the multidrug-resistance-related protein (MRP) gene family, a subfamily of the ATP-binding cassette (ABC) transporters from Arabidopsis is presented. We used cDNA sequencing and alignment-based re-annotation of genomic sequences to define the exact genic structure of all known AtMRP genes. Analysis of promoter regions suggested different induction conditions even for closely related genes. Expression analysis for the entire gene family confirmed these assumptions. Phylogenetic analysis and determination of segmental duplication in the regions of AtMRP genes revealed that the evolution of the extraordinarily high number of ABC transporter genes in plants cannot solely be explained by polyploidisation during the evolution of the Arabidopsis genome. Interestingly MRP genes from Oryza sativa L. (rice; OsMRP) show very similar genomic structures to those from Arabidopsis. Screening of large populations of T-DNA-mutagenised lines of A. thaliana resulted in the isolation of AtMRP insertion mutants. This work opens the way for the defined analysis of a multigene family of important membrane transporters whose broad variety of functions expands their traditional role as cellular detoxifiers.

Molecular characterisation of a new mutant allele of the plastid phosphoglucomutase in Arabidopsis, and complementation of the mutant with the wild-type cDNA

Abstract Screening of transposon-associated mutants of Arabidopsis thaliana for altered starch metabolism resulted in the isolation of a mutant that did not accumulate starch in any tissue or at any developmental stage (starch-free mutant, stf1). Allelism tests with known mutants showed that stf1 represents a new mutant allele of the plastid isoform of the enzyme phosphoglucomutase (PGMp). The mutation was mapped to chromosome 5. An Arabidopsis EST that showed significant homology to the cytosolic isoform of phosphoglucomutase (PGM) from maize was able to complement the mutant phenotype. The Arabidopsis EST was transcribed and translated in vitro and the protein product was efficiently imported into isolated chloroplasts and processed to its mature form. The lack of starch biosynthesis in stf1 is accompanied by the accumulation of soluble sugars. The rate of CO2 assimilation measured in individual leaves was substantially diminished only under conditions of high CO2 and low O2. Remarkably, stf1 exhibits an increase rather than a decrease in total leaf PGM activity, suggesting an induction of the cytosolic isoform(s) in the mutant. The substrate for PGM, glucose 6-phosphate, accumulated in stf1 during the day, resulting in 10-fold higher content than in the wild type at the end of the photoperiod.

Expression of the triose phosphate translocator gene from potato is light dependent and restricted to green tissues

The export of primary photosynthesis products from chloroplasts into the cytoplasm is mediated by the triose phosphate translocator. The transporter is an integral membrane protein localized at the inner envelope of chloroplasts. In order to study the expression of the major chloroplast envelope protein gene E29, which is assumed to function as the translocator, we have isolated corresponding cDNA clones from potato. A full-length clone was sequenced and shown to be highly homologous to the E29 gene from spinach. Expression on the RNA level is restricted to green tissues, is light dependent and cannot be induced by sucrose in darkness. The presence of a single-copy gene argues for the existence of different translocator systems responsible for import and export of carbohydrates in chloroplasts and amyloplasts.

Genomics of plant ABC transporters: The alphabet of photosynthetic life forms or just holes in membranes?

The large number of ABC transporters in the Arabidopsis genome was made responsible for known as well as unexpected aspects in plant development. The combination of classical transport experiments with functional genomics approaches helped unravel some of these effects. Yet questions concerning the importance of this large number of ABC transporters in eukaryotic photosynthesizing organisms remain open. Phylogenomic analyses of whole genome sequence data reveal comparable sizes and composition between algae and higher plants within this protein family. Although this indicates the significance of ABC transporters in plants, several other questions remain to be answered.