Michele Wolfe Bianchi

Arabidopsis homologs of the shaggy and GSK-3 protein kinases: molecular cloning and functional expression in Escherichia coli.

The conservation in evolution of fundamental signal transduction modules offers a means of isolating genes likely to be involved in plant development. We have amplified by PCR Arabidopsis cDNA and genomic sequences related to the product of the shaggy/zeste-white 3 (sgg) segment polarity gene of Drosophila. This regulatory protein is functionally homologous to glycogen synthase kinase-3 in mammals (GSK-3), which regulates, among others, the DNA-binding activity of the c-jun/AP1 transcription factor. Analysis of PCR products led to the identification of five genes; for two of which, corresponding full-length cDNAs, ASK-α and γ (for Arabidopsis shaggy-related protein kinase), were characterized. The encoded proteins were 70% identical to GSK-3 and sgg over the protein kinase catalytic domain and, after production in Escherichia coli, autophosphorylated mainly on threonine and serine residues, but phosphotyrosine was also detected. ASK-α and ASK-γ also phosphorylated phosphatase inhibitor-2 and myelin basic protein, on threonine and serine, respectively. The high conservation of the protein kinases of GSK-3 family, and their action at the transcriptional level, suggest that the ASK proteins have important functions in higher plants.

The RNA Binding Protein Tudor-SN Is Essential for Stress Tolerance and Stabilizes Levels of Stress-Responsive mRNAs Encoding Secreted Proteins in Arabidopsis

This study describes the role of Tudor-SN in optimal stress tolerance throughout the life cycle of Arabidopsis. It finds evidence suggestive of new mechanisms regulating the metabolism of mRNAs entering the secretory pathway. Tudor-SN (TSN) copurifies with the RNA-induced silencing complex in animal cells where, among other functions, it is thought to act on mRNA stability via the degradation of specific dsRNA templates. In plants, TSN has been identified biochemically as a cytoskeleton-associated RNA binding activity. In eukaryotes, it has recently been identified as a conserved primary target of programmed cell death–associated proteolysis. We have investigated the physiological role of TSN by isolating null mutations for two homologous genes in Arabidopsis thaliana. The double mutant tsn1 tsn2 displays only mild growth phenotypes under nonstress conditions, but germination, growth, and survival are severely affected under high salinity stress. Either TSN1 or TSN2 alone can complement the double mutant, indicating their functional redundancy. TSN accumulates heterogeneously in the cytosol and relocates transiently to a diffuse pattern in response to salt stress. Unexpectedly, stress-regulated mRNAs encoding secreted proteins are significantly enriched among the transcripts that are underrepresented in tsn1 tsn2. Our data also reveal that TSN is important for RNA stability of its targets. These findings show that TSN is essential for stress tolerance in plants and implicate TSN in new, potentially conserved mechanisms acting on mRNAs entering the secretory pathway.

The indolic compound hypaphorine produced by ectomycorrhizal fungus interferes with auxin action and evokes early responses in nonhost Arabidopsis thaliana.

Signals leading to mycorrhizal differentiation are largely unknown. We have studied the sensitivity of the root system from plant model Arabidopsis thaliana to hypaphorine, the major indolic compound isolated from the basidiomycetous fungus Pisolithus tinctorius. This fungi establishes ectomycorrhizas with Eucalyptus globulus. Hypaphorine controls root hair elongation and counteracts the activity of indole-3-acetic acid on root elongation on A. thaliana, as previously reported for the host plant. In addition, we show that hypaphorine counteracts the rapid upregulation by indole-3-acetic acid and 1-naphthalenic-acetic acid of the primary auxin-responsive gene IAA1 and induces a rapid, transient membrane depolarization in root hairs and suspension cells, due to the modulation of anion and K+ currents. These early responses indicate that components necessary for symbiosis-related differentiation events are present in the nonhost plant A. thaliana and provide tools for the dissection of the hypaphorine-auxin interaction.

ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants

Autophagosomes are the organelles responsible for macroautophagy and arise, in yeast and animals, from the sealing of a cup-shaped double-membrane precursor, the phagophore. How the phagophore is generated and grows into a sealed autophagosome is still not clear in detail, and unknown in plants. This is due, in part, to the scarcity of structurally informative, real-time imaging data of the required protein machinery at the phagophore formation site. Here we find that in intact living Arabidopsis tissue, autophagy-related protein ATG5, which is essential for autophagosome formation, is present at the phagophore site from early, sub-resolution stages and later defines a torus-shaped structure on a flat cisternal early phagophore. Movement and expansion of this structure are accompanied by the underlying endoplasmic reticulum, suggesting tight connections between the two compartments. Detailed real-time and 3D imaging of the growing phagophore are leveraged to propose a model for autophagosome formation in plants.

Identification of proteins regulated by cross-talk between drought and hormone pathways in Arabidopsis wild-type and auxin-insensitive mutants, axr1 and axr2

Ten proteins differentially regulated by progressive drought stress in Arabidopsis Columbia wild-type, axr1-3 and axr2-1auxin-insensitive mutants, were identified from internal amino acid microsequencing. These proteins fell into two categories: (i) stress-related proteins, known to be induced by rapid water stress via abscisic acid (ABA)-dependent or -independent pathways [late embryogenesis abundant (LEA)-like and heat shock cognate (HS) 70, respectively], or in response to pathogens or oxidative stress [β-1,3 glucanase (BG), annexin] and (ii) metabolic enzymes [glutamine synthetase (GS), fructokinase (Frk), caffeoyl-CoA-3-O-methyltransferase (CCoAOMT)]. The differential behaviour of these proteins highlighted a role for AXR2 and/or AXR1 in the regulation of their abundance during drought adaptation. In particular, reduced induction of RD29B, GS and annexin, and overexpression of BG2 were observed specifically in the axr1-3 mutant, which is dramatically affected in several ABA-dependent drought adaptive responses, such as drought rhizogenesis. Altogether these results indicate cross-talk between auxin- and ABA-signalling in Arabidopsis drought responses.

A Saccharomyces cerevisiae protein-serine kinase related to mammalian glycogen synthase kinase-3 and the Drosophila melanogaster gene shaggy product

The glycogen synthase kinase-3 (GSK-3) family of protein-serine kinases is implicated in the development and hormonal regulation of higher eukaryotes. GSK-3-related genes have been cloned and characterized in mammals (alpha and beta forms), Drosophila melanogaster (shaggy/zeste-white3) and Saccharomyces cerevisiae (MCK1). Using the polymerase chain reaction and primers designed to hybridize to conserved catalytic domain sequences of this family, a genomic fragment was amplified from budding yeast DNA. Genomic clones encompassing the entire reading frame were subsequently isolated and sequenced. The protein encoded by this gene, termed ScGSK-3, displays high identity with members of the GSK-3 family, sharing several structural features including a regulatory Tyr residue. A phylogenetic analysis of the catalytic domains of these protein kinases suggests that ScGSK-3 represents the bona fide homologue of GSK-3 and the shaggy product, while the related MCK1 protein kinase is encoded by a paralogous gene which originated by a gene duplication event in the yeast lineage.

Phosphatidylinositol 3-phosphate–binding protein AtPH1 controls the localization of the metal transporter NRAMP1 in Arabidopsis

Significance Metal homeostasis is essential for living organisms. Metal transporters play key roles in metal uptake and compartmentalization. The tight regulation of metal homeostasis thus depends on accurate targeting of these metal transporters. Although the main metal transporters in plants have been identified, the mechanisms involved in their trafficking are still poorly understood. This study reveals that AtPH1, a pleckstrin homology (PH) domain containing protein binding to phosphatidylinositol 3-phosphate (PI3P), controls the subcellular localization of the iron and manganese transporter AtNRAMP1. Our results further indicate that, in addition to proteins containing the FYVE and PHOX domains, proteins containing the PH domain can decode the PI3P signal in endosomal function. “Too much of a good thing” perfectly describes the dilemma that living organisms face with metals. The tight control of metal homeostasis in cells depends on the trafficking of metal transporters between membranes of different compartments. However, the mechanisms regulating the location of transport proteins are still largely unknown. Developing Arabidopsis thaliana seedlings require the natural resistance-associated macrophage proteins (NRAMP3 and NRAMP4) transporters to remobilize iron from seed vacuolar stores and thereby acquire photosynthetic competence. Here, we report that mutations in the pleckstrin homology (PH) domain-containing protein AtPH1 rescue the iron-deficient phenotype of nramp3nramp4. Our results indicate that AtPH1 binds phosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the late endosome compartment. We further show that loss of AtPH1 function leads to the mislocalization of the metal uptake transporter NRAMP1 to the vacuole, providing a rationale for the reversion of nramp3nramp4 phenotypes. This work identifies a PH domain protein as a regulator of plant metal transporter localization, providing evidence that PH domain proteins may be effectors of PI3P for protein sorting.

Folding into an autophagosome: ATG5 sheds light on how plants do it.

Autophagosomes arise in yeast and animals from the sealing of a cup-shaped double-membrane precursor, the phagophore. The concerted action of about 30 evolutionarily conserved autophagy related (ATG) proteins lies at the core of this process. However, the mechanisms allowing phagophore generation and its differentiation into a sealed autophagosome are still not clear in detail, and very little is known in plants. This is due in part to the scarcity of structurally informative, real-time imaging data of ATG proteins at the phagophore site. Among these, the ATG5 complex directs anchoring of ATG8 to the phagophore, an event required for membrane expansion. Detailed real-time and 3D imaging of ATG5, ATG8, and an ER marker at the expanding phagophore allowed us to propose a model for autophagosome formation in plants. This model implies tight connections of the growing phagophore with the outer face of the cortical endoplasmic reticulum and prompts new questions on the mechanism of autophagosome biogenesis.

Plant Genes Encoding Homologues of the SNF1 and Shaggy Protein Kinases

Critical enzymes and proteins are often regulated by the reversible addition of phosphate. The family of enzymes catalyzing this reaction is huge and diverse. There are two major classes of protein kinases: those having specificity for serine and threonine and those enzymes that phosphorylate tyrosine residues. Phosphatases reverse the effects of protein kinases and also play important roles in cellular physiology.