Eva Sundberg

Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome

Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

The Arabidopsis thaliana STYLISH1 Protein Acts as a Transcriptional Activator Regulating Auxin Biosynthesis

Biosynthesis of the plant hormone auxin must be tightly controlled. This work shows that the STYLISH1 protein of the plant species Arabidopsis thaliana plays an important role in this process by directly binding to and activating at least one of the auxin biosynthesis genes. The establishment and maintenance of auxin maxima in vascular plants is regulated by auxin biosynthesis and polar intercellular auxin flow. The disruption of normal auxin biosynthesis in mouse-ear cress (Arabidopsis thaliana) leads to severe abnormalities, suggesting that spatiotemporal regulation of auxin biosynthesis is fundamental for normal growth and development. We have shown previously that the induction of the SHORT-INTERNODES/STYLISH (SHI/STY) family member STY1 results in increased transcript levels of the YUCCA (YUC) family member YUC4 and also higher auxin levels and auxin biosynthesis rates in Arabidopsis seedlings. We have also shown previously that SHI/STY family members redundantly affect development of flowers and leaves. Here, we further examine the function of STY1 by analyzing its DNA and protein binding properties. Our results suggest that STY1, and most likely other SHI/STY members, are DNA binding transcriptional activators that target genes encoding proteins mediating auxin biosynthesis. This suggests that the SHI/STY family members are essential regulators of auxin-mediated leaf and flower development. Furthermore, the lack of a shoot apical meristem in seedlings carrying a fusion construct between STY1 and a repressor domain, SRDX, suggests that STY1, and other SHI/STY members, has a role in the formation and/or maintenance of the shoot apical meristem, possibly by regulating auxin levels in the embryo.

A method for production of interspecific hybrids within Brassiceae via somatic hybridization, using resynthesis of Brassica napus as a model

A general method for the production of somatic hybrids within Brassiceae has been developed using mesophyll protoplasts from one parent and hypocotyl protoplasts from the other. Fusion products were easily identified by their intermediate phenotype between the parental protoplasts. They could be isolated 24 h after fusion with micropipettes using a micromanipulator. At that time their frequency was about 15%. They were cultured in small volumes, 10 μl, and a plating efficiency of 14% was obtained. Hybrid calli were obtained from the fusion products, which was confirmed by isozyme analysis. Ploidy level of one hybrid shoot was determined by flow cytometric DNA analysis.

Distinct and Dynamic Auxin Activities During Reproductive Development

Flowering plants have evolved sophisticated and complicated reproductive structures to ensure optimal conditions for the next generation. Successful reproduction relies on careful timing and coordination of tissue development, which requires constant communication between these tissues. Work on flower and fruit development over the last decade places the phytohormone auxin in a key role as a master of patterning and tissue specification of reproductive organs. Although many questions still remain, it is now clear that auxin mediates its function in flowers and fruits through an integrated process of biosynthesis, transport, and signaling, as well as interaction with other hormonal pathways. In addition, the knowledge obtained so far about auxin function already allows researchers to develop tools for crop improvement and precision agriculture.

Gene transfer via somatic hybridization in plants

Abstract Successful plant protoplast culture and fusion techniques have now been developed for many crop species. This has stimulated hopes that the transfer of desirable traits between sexually incompatible species will prove to be a practical approach for the genetic improvement of crops.

Effects of parental ploidy level and genetic divergence on chromosome elimination and chloroplast segregation in somatic hybrids within Brassicaceae

SummaryChromosome and organelle segregation after the somatic hybridization of related species with different degrees of genetic divergence were studied by comparing the interspecific somatic hybrids Brassica oleracea (CC) (+) B. campestris (AA), B. napus (AACC) (+) B. oleracea (CC) B. napus (AACC) (+) B. nigra (BB) and B. napus (AACC) (+) B. juncea (AABB) with the intergeneric somatic hybrids B. napus (AACC) (+) Raphanus sativus (RR) and B. napus (AACC) (+) Eruca sativa (EE). Within each combination, some hybrids were found whose DNA content was equal to the sum of parental chromosomes, others had a relatively higher DNA content and in most of the cases, some had a relatively lower content. However, the frequency distribution in these three classes differed significantly between the combinations. A positive correlation between the frequency of hybrids with eliminated chromosomes and the genetic distance between the species in each combination was found. Furthermore, by combining species with different ploidy levels we found a significantly higher degree of chromosome elimination compared to combinations of species with the same ploidy level. In the B. napus (+) B. Nigra, B. napus (+) R. sativus and B. napus (+) E. sativa combinations chromosomes from the B, R and E genomes appeared to be preferentially sorted out, as indicated by the fact that some of the nuclear markers from these genomes were missing in 7–46% of the plants, whereas no plants were lacking B. napus nuclear markers. Fertile hybrids were found in all but the B. napus (+) R. sativus fusion combination; the latter hybrids were male sterile, but female fertile. Hybrids between the A and C genomes were more fertile than hybrids obtained between the distantly related AC and B, R or E genomes, respectively. Analysis of the chloroplast RFLP pattern revealed that chloroplasts in the B. oleracea (+) B. campestris hybrids segregated randomly. A slightly biased segregation, favouring B. napus chloroplasts, was found in the B. napus (+) B. oleracea combination, whereas B. napus chloroplasts were strongly selected for in the B. napus (+) B. juncea, B. napus (+) B. nigra, B. napus (+) R. sativus and B. napus (+) E. sativa somatic hybrids.

Fertility and chromosome stability in Brassica napus resynthesised by protoplast fusion

SummaryFertile somatic hybrids between Brassica campestris and B. oleracea have been produced by protoplast fusion. Fusion products were identified by their intermediate protoplast morphology. Heterokaryons were isolated either with micropipettes using a micromanipulator or by flow sorting. About 2% of the obtained calli differentiated to shoots. Of the shoots obtained from manually selected heterokaryons, 100% were true hybrids as confirmed by isozyme analysis while 87% of the flow sorted ones showed a hybrid pattern. Ploidy level of the hybrid plants was determined by chromosome counting and relative DNA-content analysis. The sum of the chromosome number (38) from the two fusion partners were found in 30% of the hybrids; 9% had fewer and 61% had more chromosomes. Pollen viability and seed set varied with ploidy level. Compared to natural B. napus, a pollen viability of 52%–93% and a fertility of 1%–40% was found for the somatic hybrids with normal chromosome number. Restriction enzyme analysis of chloroplast-DNA showed that either B. campestris or B. oleracea chloroplasts were present in the somatic hybrid plants. Of 11 hybrid plants 5 had the campestris and 6 had the oleracea type (1∶1 ratio).

Auxin can act independently of CRC, LUG, SEU, SPT and STY1 in style development but not apical‐basal patterning of the Arabidopsis gynoecium

Patterning of the Arabidopsis thaliana gynoecium is dependent on the localization and concentration of the plant hormone auxin and it has been previously reported that STYLISH1 (STY1) activates transcription of the auxin biosynthesis gene YUCCA4 (YUC4) and affects gynoecium development. Here, the relationship between auxin, STY1 and other regulators of gynoecium development was examined. Exogenous auxin in droplets of lanolin paste were applied to young gynoecia; auxin biosynthesis rate was measured and STY1 overexpression or chemically mediated polar auxin transport (PAT) inhibition were induced in various mutants. The style phenotype of sty1-1sty2-1 mutants was restored by exogenous application of auxin, and STY1 over-activation resulted in an elevated auxin biosynthesis rate. Both over-activation of STY1 and inhibition of PAT restored the stylar defects of several unrelated mutants, but with regard to gynoecium apical-basal patterning the mutants responded differently to inhibition of PAT. These results suggest that reduced auxin concentrations cause the sty1-1 sty2-1 phenotype, that STY1 induces auxin biosynthesis, that elevated apical auxin concentrations can compensate for the loss of several style-promoting factors, and that auxin may act downstream of, or in parallel with these during style development but is dependent on their action in apical-basal patterning.

Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens

The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.

Correlation between flow cytometric determination of nuclear DNA content and chromosome number in somatic hybrids within Brassicaceae.

SummaryFourty-one somatic hybrids from two species combinations, Brassica oleracea + B. campestris and B. napus + Eruca sativa, were analysed for chromosome number and nuclear DNA content. The DNA content was measured in a flow cytometer using two internal standards as references and when related to the chromosome number a correlation of 0.91 was found. The chromosome number of the hybrids could be determined with an accuracy of ±10% by using flow cytometry, and the smallest statistically significant difference in DNA content between two individuals was 0.23 pg DNA/cell.