Norbert Sauer

The promoter of the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene directs expression of β-glucuronidase to the phloem: Evidence for phloem loading and unloading by SUC2

The Arabidopsis thaliana (L.) Heynh. SUC2 gene encodes a plasma-membrane sucrose-H+ symporter. The DNA sequence of the SUC2 promoter has been determined. Using a translational fusion of this promoter to the N-terminus of β-glucuronidase (GUS) and the GUS histochemical assay, the tissue specificity of the SUC2 promoter was studied in Arabidopsis plants transformed with this fusion construct. The SUC2 promoter directed expression of GUS activity with high specificity to the phloem of all green tissues of Arabidopsis such as rosette leaves, stems, and sepals. During leaf development the expression of SUC2-GUS activity was first seen in the tips of young rosette leaves. In older leaves and during their concomitant sink/source transition, expression proceeded from the tips to the bases of the leaves, indicating that expression of the SUC2 sucrose-H+ symporter is tightly coupled to the source-strength of Arabidopsis leaves. Expression of SUC2-GUS activity was also seen, however, in sink tissues such as roots and developing Arabidopsis pods, suggesting that the product of the SUC2 gene might not only be important for phloem loading, but also for phloem unloading. A possible regulatory effect of carbohydrates (glucose and sucrose) on the activity of the SUC2 promoter was studied and excluded, both in excised leaves and young seedlings of transgenic Arabidopsis plants. The overall pattern of SUC2-GUS expression correlated well with that of the Arabidopsis thaliana AHA3 plasma-membrane H+ -ATPase which is also expressed in the phloem and most likely represents the primary pump generating the energy for secondary active transporters such as SUC2.

Sugar transporters in higher plants--a diversity of roles and complex regulation.

Sugar-transport proteins play a crucial role in the cell-to-cell and long-distance distribution of sugars throughout the plant. In the past decade, genes encoding sugar transporters (or carriers) have been identified, functionally expressed in heterologous systems, and studied with respect to their spatial and temporal expression. Higher plants possess two distinct families of sugar carriers: the disaccharide transporters that primarily catalyse sucrose transport and the monosaccharide transporters that mediate the transport of a variable range of monosaccharides. The tissue and cellular expression pattern of the respective genes indicates their specific and sometimes unique physiological tasks. Some play a purely nutritional role and supply sugars to cells for growth and development, whereas others are involved in generating osmotic gradients required to drive mass flow or movement. Intriguingly, some carriers might be involved in signalling. Various levels of control regulate these sugar transporters during plant development and when the normal environment is perturbed. This article focuses on members of the monosaccharide transporter and disaccharide transporter families, providing details about their structure, function and regulation. The tissue and cellular distribution of these sugar transporters suggests that they have interesting physiological roles.

Molecular physiology of higher plant sucrose transporters

Sucrose is the primary product of photosynthetic CO2 fixation that is used for the distribution of assimilated carbon within higher plants. Its partitioning from the site of synthesis to different sites of storage, conversion into other storage compounds or metabolic degradation involves various steps of cell‐to‐cell movement and transport. Many of these steps occur within symplastic domains, i.e. sucrose moves passively cell‐to‐cell through plasmodesmata. Some essential steps, however, occur between symplastically isolated cells or tissues. In these cases, sucrose is transiently released into the apoplast and its cell‐to‐cell transport depends on the activity of plasma membrane‐localized, energy dependent, H+‐symporting carrier proteins. This paper reviews the current knowledge of sucrose transporter physiology and molecular biology.

A role for sugar transporters during seed development:molecular characterization of a hexose and a sucrose carrier in fava bean seeds.

To analyze sugar transport processes during seed development of fava bean, we cloned cDNAs encoding one sucrose and one hexose transporter, designated VfSUT1 and VfSTP1, respectively. sugar uptake activity was confirmed after heterologous expression in yeast. Gene expression was studied in relation to seed development. Transcripts were detected in both vegetative and seed tissues. In the embryo, VfSUT1 and VfSTP1 mRNAs were detected only in epidermal cells, but in a different temporal and spatial pattern. VfSTP1 mRNA accumulates during the midcotyledon stage in epidermal cells covering the mitotically active parenchyma, whereas the VfSUT1 transcript was specific to outer epidermal cells showing transfer cell morphology and covering the storage parenchyma. Transfer cells developed at the contact area of the cotyledonary epidermis and the seed coat, starting first at the early cotyledon stage and subsequently spreading to the abaxial region at the late cotyledon stage. Feeding high concentrations of sugars suppressed both VfSUT1 expression and transfer cell differentiation in vitro, suggesting a control by carbohydrate availability.

Differential regulation of a hydroxyproline-rich glycoprotein gene family in wounded and infected plants.

We have characterized three different transcripts induced by fungal elicitor, wounding, or infection which encode apoproteins of cell wall hydroxyproline-rich glycoproteins involved in plant defense against infection. The proteins encoded by two of these transcripts contain a proline-rich domain involving tandem repetition of the 16-amino-acid unit Tyr3-Lys-Ser-Pro4-Ser-Pro-Ser-Pro4. The third transcript encodes a protein with a proline-rich domain involving a variant of this 16-mer canonical repeat: Tyr3-His-Ser-Pro4-Lys-His-Ser-Pro4. Each transcript is encoded by a separate gene present at single or low copy number in the haploid genome. These transcripts exhibit markedly different patterns of accumulation in different stress conditions, indicating the operation of several distinct intercellular stress signal systems in higher plants.

Glycine-rich cell wall proteins in bean: gene structure and association of the protein with the vascular system.

A single genomic clone (14 kb) isolated from bean (Phaseolus vulgaris L.) contains two genes that encode glycine‐rich proteins. These genes are present as single copies in the genome, are separated by 2.85 kb and encode transcripts of 1.8 kb and 1.0 kb respectively. The encoded proteins contain 60% glycine and have amino‐terminal signal peptides. The 1.8 kb transcript is present in young hypocotyls and in ovary tissue. Excision‐wounding transiently induced this transcript in old, but not in young hypocotyl tissue. Antibodies raised against regions of the glycine‐rich protein 1.8, expressed as a lacZ fusion protein in bacteria, react with a protein of 53 kd in a protein fraction extracted from cell walls of bean ovaries. Tissue imprints of bean ovaries treated with anti‐glycine‐rich protein antibodies showed that the glycine‐rich protein was distributed in a regular pattern of small, highly localized discrete sites. The immunoreactive regions correspond to the pattern of vascular tissue in the pod. In young hypocotyls, glycine‐rich protein is present at four pairs of discrete sites symmetrically arranged on the inner side of the vascular ring. These results suggest a close relationship between glycine‐rich proteins and development of the vascular system.

The sink-specific and stress-regulated Arabidopsis STP4 gene: enhanced expression of a gene encoding a monosaccharide transporter by wounding, elicitors, and pathogen challenge.

A cDNA for the Arabidopsis STP4 gene (for sugar transport protein 4) was isolated, and the properties of the encoded protein were studied in Schizosaccharomyces pombe. The STP4 monosaccharide H+ symporter is composed of 514 amino acids and has a calculated molecular mass of 57.1 kD. RNA gel blot analyses revealed that STP4 is expressed primarily in roots and flowers of Arabidopsis. This was shown in more detail with STP4 promoter-beta-glucuronidase (GUS) plants yielding strong STP4-driven GUS activity in root tips and anthers. Wounding of plants transformed with STP4-GUS constructs resulted in a rapid increase in GUS activity in cells directly adjacent to the lesion. This was confirmed by RNase protection analyses in Arabidopsis wild-type plants showing a strong, wound-induced increase in STP4 mRNA levels. STP4 expression was induced rapidly in suspension-cultured Arabidopsis cells that were treated with the Pseudomonas syringae elicitor or with chitin or in Arabidopsis plants that were exposed to fungal attacks. Our data suggest that the role of STP4 is to catalyze monosaccharide import into classic sinks, such as root tips and anthers, and, most importantly, to meet the increased carbohydrate demand of cells responding to environmental stress.

Callose Biosynthesis Regulates Symplastic Trafficking during Root Development

Plant cells are connected through plasmodesmata (PD), membrane-lined channels that allow symplastic movement of molecules between cells. However, little is known about the role of PD-mediated signaling during plant morphogenesis. Here, we describe an Arabidopsis gene, CALS3/GSL12. Gain-of-function mutations in CALS3 result in increased accumulation of callose (β-1,3-glucan) at the PD, a decrease in PD aperture, defects in root development, and reduced intercellular trafficking. Enhancement of CALS3 expression during phloem development suppressed loss-of-function mutations in the phloem abundant callose synthase, CALS7 indicating that CALS3 is a bona fide callose synthase. CALS3 alleles allowed us to spatially and temporally control the PD aperture between plant tissues. Using this tool, we are able to show that movement of the transcription factor SHORT-ROOT and microRNA165 between the stele and the endodermis is PD dependent. Taken together, we conclude that regulated callose biosynthesis at PD is essential for cell signaling.

The Monosaccharide Transporter Gene, AtSTP4, and the Cell-Wall Invertase, Atβfruct1, Are Induced in Arabidopsis during Infection with the Fungal Biotroph Erysiphe cichoracearum

Powdery mildew fungi are biotrophic pathogens that form a complex interface, the haustorium, between the host plant and the parasite. The pathogen acts as an additional sink, competing with host sinks, resulting in considerable modification of photoassimilate production and partitioning within the host tissue. Here, we examine the factors that may contribute to these changes. We show for the first time in one biotrophic interaction (Arabidopsis/Erysiphe cichoracearum) all of the following responses: Glc uptake in host tissues is enhanced after fungal infection; this coincides with the induction of expression of the monosaccharide transporter gene, Arabidopsis sugar transport protein 4 (AtSTP4), in infected leaves; invertase activity and transcript levels for a cell wall invertase, Atβfruct1, increase substantially in Arabidopsis during attack by this pathogen. Before infection, Arabidopsis plants transformed with an AtSTP4 promoter-β-glucuronidase construct show expression mainly in sink tissues such as roots; after infection, AtSTP4 expression is induced in the mature leaves and increases over the 6-d time period. Sections of infected leaves stained for β-glucuronidase show that AtSTP4 expression is not confined to infected epidermal cells but is also evident in a wider range of cells, including those of the vascular tissue. The results are discussed in relation to the possible coordinated expression of hexose transporters and cell wall invertase in the host response to powdery mildew infection.

A Versatile Monosaccharide Transporter That Operates in the Arbuscular Mycorrhizal Fungus Glomus sp Is Crucial for the Symbiotic Relationship with Plants

Carbon allocation to arbuscular mycorrhizal fungi is the reward that plants offer their symbiotic partners in exchange for mineral nutrients. This study identifies a monosaccharide transporter that plays a key role in this process. For more than 400 million years, plants have maintained a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi. This evolutionary success can be traced to the role of these fungi in providing plants with mineral nutrients, particularly phosphate. In return, photosynthates are given to the fungus, which support its obligate biotrophic lifestyle. Although the mechanisms involved in phosphate transfer have been extensively studied, less is known about the reciprocal transfer of carbon. Here, we present the high-affinity Monosaccharide Transporter2 (MST2) from Glomus sp with a broad substrate spectrum that functions at several symbiotic root locations. Plant cell wall sugars can efficiently outcompete the Glc uptake capacity of MST2, suggesting they can serve as alternative carbon sources. MST2 expression closely correlates with that of the mycorrhiza-specific Phosphate Transporter4 (PT4). Furthermore, reduction of MST2 expression using host-induced gene silencing resulted in impaired mycorrhiza formation, malformed arbuscules, and reduced PT4 expression. These findings highlight the symbiotic role of MST2 and support the hypothesis that the exchange of carbon for phosphate is tightly linked. Unexpectedly, we found that the external mycelium of AM fungi is able to take up sugars in a proton-dependent manner. These results imply that the sugar uptake system operating in this symbiosis is more complex than previously anticipated.