Characterization of ScORK28, a transmembrane functional protein receptor kinase predominantly expressed in ovaries from the wild potato species Solanum chacoense

Solanum chacoense ovule receptor kinase 28 (ScORK28) was found among 30 receptor kinases from an ovule cDNA library enriched for weakly expressed mRNAs. This LRR-RLK displayed high level of tissue specificity at the RNA and protein levels and was predominantly expressed in female reproductive tissues. Protein expression analyses in planta revealed that ScORK28 was N-glycosylated and ScORK28::GFP fusion analyses showed that it was localized at the plasma membrane. Bacterial expression of ScORK28 catalytic domain followed by kinase activity assays revealed that ScORK28 is an active Mg2+-dependent protein kinase and that the juxtamembrane domain is necessary for kinase activity.     

The MIDASIN and NOTCHLESS genes are essential for female gametophyte development in Arabidopsis thaliana

Female gametophyte development in Arabidopsis thaliana follows a well-defined program that involves many fundamental cellular processes. In this study, we report the involvement of the Arabidopsis thaliana MIDASIN1 (AtMDN1) gene during female gametogenesis through the phenotypic characterization of plants heterozygous for an insertional mdn1 mutant allele. The MDN1 yeast ortholog has previously been shown to encode a non-ribosomal protein involved in the maturation and assembly of the 60S ribosomal subunit. Heterozygous MDN1/mdn1 plants were semisterile and mdn1 allele transmission through the female gametophyte was severely affected. Development of mdn1 female gametophyte was considerably delayed compared to their wild-type siblings. However, delayed mdn1 female gametophytes were able to reach maturity and a delayed pollination experiment showed that a small proportion of the female gametophytes were functional. We also report that the Arabidopsis NOTCHLESS (AtNLE) gene is also required for female gametogenesis. The NLE protein has been previously shown to interact with MDN1 and to be also involved in 60S subunit biogenesis. The introduction of an AtNLE-RNA interference construct in Arabidopsis led to semisterility defects. Defective female gametophytes were mostly arrested at the one-nucleate (FG1) developmental stage. These data suggest that the activity of both AtMDN1 and AtNLE is essential for female gametogenesis progression.     

Loss of ovule identity induced by overexpression of the fertilization-related kinase 2 (ScFRK2), a MAPKKK from Solanum chacoense

In order to gain information about protein kinases acting during plant fertilization and embryogenesis, a reverse genetic approach was used to determine the role of protein kinases expressed in reproductive tissues. Two cDNA clones named ScFRK1 and ScFRK2 (Solanum chacoense fertilization-related kinase 1 and 2) were isolated from an expressed sequence tag (EST) library normalized for weakly expressed genes in fertilized ovaries. These showed significant sequence similarities to members of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. RNA gel blot and RNA in situ hybridization analyses confirmed the strong up-regulation of ScFRK2 in ovules after fertilization. In addition, ScFRK2 mRNAs accumulate during early ovule development in the megasporocyte and in the integument of developing ovules. Overexpression of ScFRK2 led to the production of fruits with a severely reduced number of seeds. The seeds that were produced also exhibited developmental retardation. Analysis of ovaries prior to fertilization showed that the seedless phenotype was caused by a homeotic conversion of ovules into carpel-like structures. The present observations are consistent with the role of ScFRK2 in pre- and post-fertilization events. Furthermore, overexpression of ScFRK2 led to changes in the expression of the class D floral homeotic gene ScFBP11, suggesting that the ScFRK2 kinase may interact, directly or indirectly, with the FBP7/11 pathway that directs establishment of ovule identity.     

ScORK17, a transmembrane receptor-like kinase predominantly expressed in ovules is involved in seed development

The mRNA expression of the Solanum chacoense Ovule Receptor Kinase 17 (ScORK17), a receptor kinase of the LRR-VI subfamily, is highly specific to the female reproductive tissues. No LRR-VI subfamily members in any plant species have yet been attributed a function. A phylogenetic tree inferred using the kinase domain of LRR-VI subfamily members separated the family into two clades: one containing an average of 8.2 LRR per protein and a second clade containing an average of 2.7. In situ hybridization analyses showed that the ScORK17 signal was mainly detected in the single ovule integument and in the endothelium. Transient expression analysis also revealed that ScORK17 was N-glycosylated in planta. Overexpression of ScORK17 in S. chacoense did not produce plants with an altered phenotype. However, when heterologous transformation was performed with a full-length ScORK17 clone in A. thaliana, the resulting transgenic plants showed reduced seed set, mainly due to aberrant embryo sac development, thus supporting a developmental role for ScORK17 in ovule and seed development.     

The Egg apparatus 1 gene from maize is a member of a large gene family found in both monocots and dicots

The maize ZmEA1 protein was recently postulated to be involved in short-range pollen tube guidance from the embryo sac. To date, EA1-like sequences had only been identified in monocot species. Using a more conserved C-terminal motif found in the monocot species, numerous ZmEA1-like sequences were retrieved in EST databases from dicot species, as well as from unannotated genomic sequences of Arabidopsis thaliana. RT-PCR analyses were produced for these unannotated genes and showed that these were indeed expressed genes. Further structural and phylogenetic analyses revealed that all members of the EA1-like (EAL) gene family shared a conserved 27–29 amino acid motif, termed the EA box near the C-terminal end, and appear to be secretory proteins. Therefore, the EA box proteins defines a new class of small secretory proteins, some of which being possibly involved in pollen tube guidance. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Ectopic expression of a wood-abundant expansin PttEXPA1 promotes cell expansion in primary and secondary tissues in aspen

Expansins are primary agents inducing cell wall extension, and are therefore obvious targets in biotechnological applications aimed at the modification of cell size in plants. In trees, increased fibre length is a goal of both breeding and genetic engineering programmes. We used an α-expansin Ptt EXPA1 that is highly abundant in the wood-forming tissues of hybrid aspen ( Populus tremula L. ×  P. tremuloides Michx.) to evaluate its role in fibre elongation and wood cell development. Ptt EXPA1 belongs to Subfamily A of α-expansins that have conserved motifs at the N- and C-termini of the mature protein. When PttEXPA1 was over-expressed in aspen, an extract of the cell wall-bound proteins of the transgenic plants exhibited an increased expansin activity on cellulose–xyloglucan composites in vitro , indicating that Ptt EXPA1 is an active expansin. The transgenic lines exhibited increased stem internode elongation and leaf expansion, and larger cell sizes in the leaf epidermis, indicating that Ptt EXPA1 protein is capable of increasing the growth of these organs by enhancing cell wall expansion in planta . Wood cell development was also modified in the transgenic lines, but the effects were different for vessel elements and fibres, the two main cell types of aspen wood. Ptt EXPA1 stimulated fibre, but not vessel element, diameter growth, and marginally increased vessel element length, but did not affect fibre length. The observed differences in responsiveness to expansin of these cell types are discussed in the light of differences in their growth strategies and cell wall composition.