Beatrix M. Horvath

The ENOD12 gene product is involved in the infection process during the pea-rhizobium interaction

The pea cDNA clone pPsENOD12 represents a gene involved in the infection process during Pisum sativum L.-Rhizobium leguminosarum bv. viciae symbiosis. The ENOD12 protein is composed of pentapeptides containing two hydroxyprolines. The expression of the ENOD12 gene is induced in cells through which the infection thread is migrating, but also in cells that do not yet contain an infection thread. Soluble compounds from Rhizobium are involved in eliciting ENOD12 gene expression. Rhizobium common and host-specific nodulation genes are essential for the production of these compounds. Two ENOD12 genes are expressed in nodules and in stem tissue of uninoculated plants. The gene represented by the cloned ENOD12 mRNA is also expressed in flowers, but a different transcription start may be used.

EBP1 regulates organ size through cell growth and proliferation in plants

Plant organ size shows remarkable uniformity within species indicating strong endogenous control. We have identified a plant growth regulatory gene, functionally and structurally homologous to human EBP1. Plant EBP1 levels are tightly regulated; gene expression is highest in developing organs and correlates with genes involved in ribosome biogenesis and function. EBP1 protein is stabilised by auxin. Elevating or decreasing EBP1 levels in transgenic plants results in a dose‐dependent increase or reduction in organ growth, respectively. During early stages of organ development, EBP1 promotes cell proliferation, influences cell‐size threshold for division and shortens the period of meristematic activity. In postmitotic cells, it enhances cell expansion. EBP1 is required for expression of cell cycle genes; CyclinD3;1, ribonucleotide reductase 2 and the cyclin‐dependent kinase B1;1. The regulation of these genes by EBP1 is dose and auxin dependent and might rely on the effect of EBP1 to reduce RBR1 protein level. We argue that EBP1 is a conserved, dose‐dependent regulator of cell growth that is connected to meristematic competence and cell proliferation via regulation of RBR1 level.

Host-specific regulation of nodulation genes in Rhizobium is mediated by a plant-signal, interacting with the nodD gene product

We have identified a nodD gene from the wide host‐range Rhizobium strain MPIK3030 (termed nodD1) which is essential for nodulation on Macroptilium atropurpureum (siratro). Experiments with nodA–lacZ gene fusions demonstrate that the MPIK3030 nodD1 regulates expression of the nodABC genes. Additionally, we used nodC–lacZ fusions of Rhizobium meliloti to show that the MPIK3030 nodD1 gene induces expression of these fusions by interacting with plant factors from siratro and from the non‐host Medicago sativa (alfalfa). The R. meliloti nodD genes, however, only interact with alfalfa exudate. In line with these results, no complementation of MPIK3030 nodD1 mutants could be obtained on siratro with the R. meliloti nodD genes, while the MPIK3030 nodD1 can complement nodD mutants of R. meliloti on alfalfa. Furthermore, R. meliloti transconjugants harbouring the MPIK3030 nodD1 efficiently nodulate the illegitimate host siratro. When compared with other nodD sequences, the amino acid sequence of the MPIK3030 nodD1 shows a conserved aminoterminus, whereas the carboxy‐terminus of the putative gene product diverges considerably. Studies on a chimeric MPIK3030/R. meliloti nodD gene indicates that the carboxy‐terminal region is responsible for the interaction with plant factor(s) and may have evolved in different rhizobia specifically to interact with plant–host factors.

Naturally occurring allele diversity allows potato cultivation in northern latitudes

Potato (Solanum tuberosum L.) originates from the Andes and evolved short-day-dependent tuber formation as a vegetative propagation strategy. Here we describe the identification of a central regulator underlying a major-effect quantitative trait locus for plant maturity and initiation of tuber development. We show that this gene belongs to the family of DOF (DNA-binding with one finger) transcription factors and regulates tuberization and plant life cycle length, by acting as a mediator between the circadian clock and the StSP6A mobile tuberization signal. We also show that natural allelic variants evade post-translational light regulation, allowing cultivation outside the geographical centre of origin of potato. Potato is a member of the Solanaceae family and is one of the world’s most important food crops. This annual plant originates from the Andean regions of South America. Potato develops tubers from underground stems called stolons. Its equatorial origin makes potato essentially short-day dependent for tuberization and potato will not make tubers in the long-day conditions of spring and summer in the northern latitudes. When introduced in temperate zones, wild material will form tubers in the course of the autumnal shortening of day-length. Thus, one of the first selected traits in potato leading to a European potato type is likely to have been long-day acclimation for tuberization. Potato breeders can exploit the naturally occurring variation in tuberization onset and life cycle length, allowing varietal breeding for different latitudes, harvest times and markets.

Arabidopsis E2FA stimulates proliferation and endocycle separately through RBR-bound and RBR-free complexes

Post‐embryonic growth in plants depends on the continuous supply of undifferentiated cells within meristems. Proliferating cells maintain their competence for division by active repression of differentiation and the associated endocycle entry. We show by upregulation and downregulation of E2FA that it is required for maintaining proliferation, as well as for endocycle entry. While E2FB–RBR1 (retinoblastoma‐related protein 1) complexes are reduced after sucrose addition or at elevated CYCD3;1 levels, E2FA maintains a stable complex with RBR1 in proliferating cells. Chromatin immunoprecipitation shows that RBR1 binds in the proximity of E2F promoter elements in CCS52A1 and CSS52A2 genes, central regulators for the switch from proliferation to endocycles. Overexpression of a truncated E2FA mutant (E2FAΔRB) lacking the RBR1‐binding domain interferes with RBR1 recruitment to promoters through E2FA, leading to decreased meristem size in roots, premature cell expansion and hyperactivated endocycle in leaves. E2F target genes, including CCS52A1 and CCS52A2, are upregulated in E2FAΔRB and e2fa knockout lines. These data suggest that E2FA in complex with RBR1 forms a repressor complex in proliferating cells to inhibit premature differentiation and endocycle entry. Thus, E2FA regulates organ growth via two distinct, sequentially operating pathways.

Pea lines carrying sym1 or sym2 can be nodulated by Rhizobium strains containing nodX ; sym1 and sym2 are allelic

In wild pea varieties two genes, sym1 and sym2, have been identified that cause resistance to European Rhizobium leguminosarum bv. viciae (Rlv) strains. The sym2 gene has previously been studied in some detail and it was shown that the additional nodulation gene nodX is sufficient to overcome the sym2 controlled nodulation resistance. Here we characterize the sym1 gene. We show that the resistance conferred by sym1 can be overcome by the introduction of nodX in European Rlv strains, indicating that sym1 just as sym2 is involved in Nod factor recognition. Both sym1 and sym2 display a recessive or dominant nature depending on the Rlv strain used for inoculation. Furthermore, introgression lines containing either sym1 or sym2 are able to form nodules with Rlv strain 248 at 26°C, but not at 18°C, indicating that both sym1 and sym2 have a temperature sensitive nature. sym2 was mapped on the pea RFLP map. We found that sym1 maps in the same region of chromosome 1 as sym2. By crossing sym1 and sym2 containing introgression lines we demonstrate that sym1 and sym2 are allelic.

Balancing act: matching growth with environment by the TOR signalling pathway

One of the most fundamental aspects of growth in plants is its plasticity in relation to fluctuating environmental conditions. Growth of meristematic cells relies predominantly on protein synthesis, one of the most energy-consuming activities in cells, and thus is tightly regulated in accordance with the available nutrient and energy supplies. The Target of Rapamycin (TOR) signalling pathway takes a central position in this regulation. The core of the TOR signalling pathway is conserved throughout evolution, and can be traced back to the last eukaryotic common ancestor. In plants, a single complex constitutes the TOR signalling pathway. Manipulating the components of the TOR complex in Arabidopsis highlighted its common role as a major regulator of protein synthesis and metabolism, that is also involved in other biological functions such as cell-wall integrity, regulation of cell proliferation, and cell size. TOR, as an integral part of the auxin signalling pathway, connects hormonal and nutrient pathways. Downstream of TOR, S6 kinase and the ribosomal S6 protein have been shown to mediate several of these responses, although there is evidence of other complex non-linear TOR signalling pathway structures.

Expression analysis of a family of nsLTP genes tissue specifically expressed throughout the plant and during potato tuber life cycle.

Non-specific lipid-transfer proteins (nsLTPs) are capable of binding lipid compounds in plant tissues and are coded by the nsLTP genes. Here, we present the analysis of expression of a family of potato (Solanum tuberosum)nsLTP genes that express throughout the developing plant in a highly tissue-specific manner. Three transcript-derived fragments were isolated using an amplified restriction fragment polymorphism-derived technique for RNA fingerprinting that show homology to plant nsLTP genes. These transcript-derived fragments displayed modulated expression profiles related to the development of new tissues, with a peak of transcription around the time of tuberization and just prior to sprout development, at dormancy breakage. In addition, a homologous family of expressed sequence tags was identified whose individual members could be classified according to their tissue specificity. Two subgroups of expressed sequence tags were found to express during tuber life cycle. To study the regulation of potato nsLTP genes, two putative potato nsLTP promoters were isolated and their expression was studied using promoter-marker-gene fusions. The results showed that one of the two promoters directed a highly specific pattern of expression detected in the phloem surrounding the nodes of young plants and in the same tissue of tuber related organs, whereas the second putative promoter showed little tissue or organ specificity. This difference in expression is likely due to a 331-bp insertion present in the tissue-specific promoter.

Isolation and functional characterization of a stolon specific promoter from potato (Solanum tuberosum L.).

In the search for time- and tissue-specific promoters an RNA fingerprinting technique called cDNA-AFLP was used. A transcript derived fragment (TDF511) was isolated which showed high similarity to alcohol dehydrogenases. The gene corresponding to this TDF, named Stgan, is likely to be involved in biosynthesis or breakdown of compounds affecting gibberellic acid (GA) levels in the plant [Plant J. 25(6) (2001) 595]. In this article the isolation and characterization of a Stgan promoter region is reported. The promoter region of this gene was fused to a reporter gene encoding beta-glucuronidase (GUS) and introduced in potato plants. GUS staining was detected uniquely in stolon tips and nodes. RNA in situ hybridization experiments revealed that this gene was specifically expressed in parenchyma cells, in the stolon cortex. Comparison of this promoter sequence with several promoter databases resulted in the identification of several potential binding sites for transcription factors. From the in vitro-culture experiments Stgan transcription appears to be induced by long days, sucrose and different hormones such as gibberellic acid, ancymidol, ethylene and cytokinins.

Isolation of a Gene Encoding a Copper Chaperone for the Copper/Zinc Superoxide Dismutase and Characterization of Its Promoter in Potato

Gene expression during the potato (Solanum tuberosum) tuber lifecycle was monitored by cDNA-amplified fragment-length polymorphism, and several differentially expressed transcript-derived fragments were isolated. One fragment, named TDFL431, showed high homology to a copper (Cu) chaperone for Cu/zinc superoxide dismutase (CCS). The Ccs protein is responsible for the delivery of Cu to the Cu/zinc superoxide dismutase enzyme. The potato CCS (StCCS) full-length gene was isolated, and its sequence was compared with CCSs from other species. The promoter region of this gene was isolated, fused to the firefly luciferase coding sequence, and used for transformation of potato plants. The highest level of StCCS-luciferase expression was detected in the cortex of stem (like) tissues, such as stem nodes, stolons, and tubers; lower levels were detected in roots and flowers. The StCCS promoter contains regions highly homologous to several plant cis-acting elements. Three of them are related to auxin response, whereas four others are related to response to various stresses. Induction of the StCCS promoter was analyzed on 18 media, differing in hormone, sugar, and Cu content. StCCS expression was induced by auxin, gibberellins (GA4 + 7), fructose, sucrose, and glucose and was inhibited by relatively high concentrations of Cu.