Publications - Stress and Develop Biology

The putative two‐pore Ca2+ channel TPC1 has been suggested to be involved in responses to abiotic and biotic stresses. We show that AtTPC1 co‐localizes with the K+‐selective channel AtTPK1 in the vacuolar membrane. Loss of AtTPC1 abolished Ca2+‐activated slow vacuolar (SV) currents, which were increased in AtTPC1 ‐over‐expressing Arabidopsis compared to the wild‐type. A Ca2+‐insensitive vacuolar cation channel, as yet uncharacterized, could be resolved in tpc1‐2 knockout plants. The kinetics of ABA‐ and CO2‐induced stomatal closure were similar in wild‐type and tpc1‐2 knockout plants, excluding a role of SV channels in guard‐cell signalling in response to these physiological stimuli. ABA‐, K+‐, and Ca2+‐dependent root growth phenotypes were not changed in tpc1‐2 compared to wild‐type plants. Given the permeability of SV channels to mono‐ and divalent cations, the question arises as to whether TPC1 in vivo represents a pathway for Ca2+ entry into the cytosol. Ca2+ responses as measured in aequorin‐expressing wild‐type, tpc1‐2 knockout and TPC1 ‐over‐expressing plants disprove a contribution of TPC1 to any of the stimulus‐induced Ca2+ signals tested, including abiotic stresses (cold, hyperosmotic, salt and oxidative), elevation in extracellular Ca2+ concentration and biotic factors (elf18, flg22). In good agreement, stimulus‐ and Ca2+‐dependent gene activation was not affected by alterations in TPC1 expression. Together with our finding that the loss of TPC1 did not change the activity of hyperpolarization‐activated Ca2+‐permeable channels in the plasma membrane, we conclude that TPC1, under physiological conditions, functions as a vacuolar cation channel without a major impact on cytosolic Ca2+ homeostasis.

Natural variation of plant pathogen resistance is often quantitative. This type of resistance can be genetically dissected in quantitative resistance loci (QRL). To unravel the molecular basis of QRL in potato (Solanum tuberosum), we employed the model plant Arabidopsis thaliana for functional analysis of natural variants of potato allene oxide synthase 2 (StAOS2). StAOS2 is a candidate gene for QRL on potato chromosome XI against the oömycete Phytophthora infestans causing late blight, and the bacterium Erwinia carotovora ssp. atroseptica causing stem black leg and tuber soft rot, both devastating diseases in potato cultivation. StAOS2 encodes a cytochrome P450 enzyme that is essential for biosynthesis of the defense signaling molecule jasmonic acid. Allele non-specific dsRNAi-mediated silencing of StAOS2 in potato drastically reduced jasmonic acid production and compromised quantitative late blight resistance. Five natural StAOS2 alleles were expressed in the null Arabidopsis aos mutant under control of the Arabidopsis AOS promoter and tested for differential complementation phenotypes. The aos mutant phenotypes evaluated were lack of jasmonates, male sterility and susceptibility to Erwinia carotovora ssp. carotovora. StAOS2 alleles that were associated with increased disease resistance in potato complemented all aos mutant phenotypes better than StAOS2 alleles associated with increased susceptibility. First structure models of ‘quantitative resistant’ versus ‘quantitative susceptible’ StAOS2 alleles suggested potential mechanisms for their differential activity. Our results demonstrate how a candidate gene approach in combination with using the homologous Arabidopsis mutant as functional reporter can help to dissect the molecular basis of complex traits in non model crop plants.

In plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two‐hybrid screening we have found three previously uncharacterized receptor‐like protein kinases to be Rop GTPase‐interacting molecules: a cysteine‐rich receptor kinase, named NCRK, and two receptor‐like cytosolic kinases from the Arabidopsis RLCK‐VIb family, named RBK1 and RBK2. Uniquely for Rho‐family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP‐bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro , suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co‐localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single‐copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK‐VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2 . We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross‐talk between Rop GTPases and specific receptor‐like kinases through direct molecular interaction.

0

The plant life cycle includes diploid sporophytic and haploid gametophytic generations. Female gametophytes (embryo sacs) in higher plants are embedded in specialized sporophytic structures (ovules). Here, we report that two closely related mitogen-activated protein kinases in Arabidopsis thaliana, MPK3 and MPK6, share a novel function in ovule development: in the MPK6 mutant background, MPK3 is haplo-insufficient, giving female sterility when heterozygous. By contrast, in the MPK3 mutant background, MPK6 does not show haplo-insufficiency. Using wounding treatment, we discovered gene dosage–dependent activation of MPK3 and MPK6. In addition, MPK6 activation is enhanced when MPK3 is null, which may help explain why mpk3−/− mpk6+/− plants are fertile. Genetic analysis revealed that the female sterility of mpk3+/− mpk6−/− plants is a sporophytic effect. In mpk3+/− mpk6−/− mutant plants, megasporogenesis and megagametogenesis are normal and the female gametophyte identity is correctly established. Further analysis demonstrates that the mpk3+/− mpk6−/− ovules have abnormal integument development with arrested cell divisions at later stages. The mutant integuments fail to accommodate the developing embryo sac, resulting in the embryo sacs being physically restricted and female reproductive failure. Our results highlight an essential function of MPK3 and MPK6 in promoting cell division in the integument specifically during ovule development.

Zinc is an essential micronutrient, and yet it can be toxic when present in excess. Zinc acquisition and distribution are dependent on tightly controlled transport of Zn2+ ions. Schizosaccharomyces pombe represents a second eukaryotic model to study cellular metal homeostasis. In several ways its micronutrient metabolism is fundamentally different from Saccharomyces cerevisiae. We identified the first Zn2+-uptake system in S. pombe and named it SpZrt1. Knock-out strains for all three ZIP (Zrt, Irt-like protein) transporters in fission yeast were constructed. Only zrt1Δ cells were unable to grow at low Zn2+ and showed reduced65Zn2+ uptake. Elemental profiles revealed a strong decrease in zinc accumulation. Cd2+ ions inhibited uptake but Fe2+ or Mn2+ did not. Both mRNA abundance and protein amount are tightly regulated. Zrt1 activity is rapidly shut down upon transfer of zinc-deficient cells to zinc-replete conditions. In cells lacking Zhf, a transporter mediating endoplasmic reticulum storage of zinc, this response is about 100-fold more sensitive. Thus, removal of excess of zinc from the cytosol is largely Zhf dependent. Moreover, cells deficient for both transporters are no longer able to adjust to changing external Zn2+ concentrations. Optimal growth is restricted to a narrow range of Zn2+ concentrations, illustrating the fine balance between micronutrient deficiency and toxicity.