J-domain cochaperones confer functional specificity to their heat shock protein (HSP)70 partner by recruiting it to specific substrate proteins. To gain insight into the functions of plastidic HSP70s, we searched in Chlamydomonas databases for expressed sequence tags that potentially encode chloroplast-targeted J-domain cochaperones. Two such cDNAs were found: the encoded J-domain proteins were named chloroplast DnaJ homolog 1 and 2 (CDJ1 and CDJ2). CDJ2 was shown to interact with a ϳ28-kDa protein that by mass spectrometry was identified as the vesicle-inducing protein in plastids 1 (VIPP1). In fractionation experiments, CDJ2 was detected almost exclusively in the stroma, whereas VIPP1 was found in low-density membranes, thylakoids, and in the stroma. Coimmunoprecipitation and mass spectrometry analyses identified stromal HSP70B as the major protein interacting with soluble VIPP1, and, as confirmed by cross-linking data, as chaperone partner of CDJ2. In blue native-PAGE of soluble cell extracts, CDJ2 and VIPP1 comigrated in complexes of Ͼ Ͼ669, ϳ150, and perhaps ϳ300 kDa. Our data suggest that CDJ2, presumably via coiled-coil interactions, binds to VIPP1 and presents it to HSP70B in the ATP state. Our findings and the previously reported requirement of VIPP1 for the biogenesis of thylakoid membranes point to a role for the HSP70B/CDJ2 chaperone pair in this process. INTRODUCTIONChaperones of the heat shock protein (Hsp)70 family belong to the most conserved proteins known. Except for some Archaea, Hsp70s are found in all known organisms and are present in every compartment of the eukaryotic cell (Bukau and Horwich, 1998). Principally, Hsp70s consist of an Nterminal ATPase domain and a C-terminal substrate-binding domain. ATP hydrolysis at the ATPase domain regulates substrate binding and release. Substrate proteins recognized by Hsp70 expose hydrophobic regions, a characteristic feature not only of nonnative proteins, but also of native Hsp70 substrates. Binding of Hsp70 to hydrophobic regions prevents the formation of aggregates. In addition, the intrinsic secondary amide peptide bond cis-trans isomerase activity recently detected for DnaK (the Hsp70 of Escherichia coli) may introduce conformational changes to bound substrates that eventually allow nonnative proteins to reconvert to the native state (Schiene-Fischer et al., 2002). Thus, Hsp70s play a major role in the folding of nascent chains and in the renaturation of nonnative proteins that have accumulated during stress situations such as heat shock (Frydman, 2001). However, they also are involved in many highly specialized functions such as the regulation of the general stress response (Tomoyasu et al., 1998), the uncoating of clathrincoated vesicles (Ungewickell et al., 1995), or the translocation of proteins across membranes (Kang et al., 1990).Specificity of Hsp70 function is mediated largely by its cochaperones, of which the J-domain cochaperones represent an important class. J-domain cochaperones contain a highly conserved J-domain that is responsibl...

Polycomb-repressive complexes (PRCs) ensure the correct spatiotemporal expression of numerous key developmental regulators. Despite their pivotal role, how PRCs are recruited to specific targets remains largely unsolved, particularly in plants. Here we show that the Arabidopsis ASYMMETRIC LEAVES complex physically interacts with PRC2 and recruits this complex to the homeobox genes BREVIPEDICELLUS and KNAT2 to stably silence these stem cell regulators in differentiating leaves. The recruitment mechanism resembles the Polycomb response element-based recruitment of PRC2 originally defined in flies and provides the first such example in plants. Combined with recent studies in mammals, our findings reveal a conserved paradigm to epigenetically regulate homeobox gene expression during development.

SummaryWe report here on the characterization of heat shock factor 1 (HSF1), encoded by one of two HSF genes identified in the genome of Chlamydomonas reinhardtii. Chlamydomonas HSF1 shares features characteristic of class A HSFs of higher plants. HSF1 is weakly expressed under non-stress conditions and rapidly induced by heat shock. Heat shock also resulted in hyperphosphorylation of HSF1, and the extent of phosphorylation correlated with the degree of induction of heat shock genes, suggesting a role for phosphorylation in HSF1 activation. HSF1, like HSFs in yeasts, forms high-molecular-weight complexes, presumably trimers, under nonstress, stress and recovery conditions. Immunoprecipitation of HSF1 under these conditions led to the identification of cytosolic HSP70A as a protein constitutively interacting with HSF1. Strains in which HSF1 was strongly under-expressed by RNAi were highly sensitive to heat stress.14 C-labelling of nuclear-encoded proteins under heat stress revealed that synthesis of members of the HSP100, HSP90, HSP70, HSP60 and small HSP families in the HSF1-RNAi strains was dramatically reduced or completely abolished. This correlated with a complete loss of HSP gene induction at the RNA level. These data suggest that HSF1 is a key regulator of the stress response in Chlamydomonas.

Flattened leaf architecture is not a default state but depends on positional information to precisely coordinate patterns of cell division in the growing primordium. This information is provided, in part, by the boundary between the adaxial (top) and abaxial (bottom) domains of the leaf, which are specified via an intricate gene regulatory network whose precise circuitry remains poorly defined. Here, we examined the contribution of the ASYMMETRIC LEAVES (AS) pathway to adaxial-abaxial patterning in Arabidopsis thaliana and demonstrate that AS1-AS2 affects this process via multiple, distinct regulatory mechanisms. AS1-AS2 uses Polycomb-dependent and -independent mechanisms to directly repress the abaxial determinants MIR166A, YABBY5, and AUXIN RESPONSE FACTOR3 (ARF3), as well as a nonrepressive mechanism in the regulation of the adaxial determinant TAS3A. These regulatory interactions, together with data from prior studies, lead to a model in which the sequential polarization of determinants, including AS1-AS2, explains the establishment and maintenance of adaxial-abaxial leaf polarity. Moreover, our analyses show that the shared repression of ARF3 by the AS and trans-acting small interfering RNA (ta-siRNA) pathways intersects with additional AS1-AS2 targets to affect multiple nodes in leaf development, impacting polarity as well as leaf complexity. These data illustrate the surprisingly multifaceted contribution of AS1-AS2 to leaf development showing that, in conjunction with the ta-siRNA pathway, AS1-AS2 keeps the Arabidopsis leaf both flat and simple.

The aim of this work was to identify cis-regulatory sequences within the Chlamydomonas HSP70A promoter that mediate its stimulatory effect on the expression of downstream promoters. For this, we deleted/mutated the HSP70A promoter and, using a new assay, quantified its stimulatory effect. Our results indicate that the effect is mediated largely by heat shock elements and the TATA box.Transgenic approaches with Chlamydomonas often suffer from the low percentage of transformants that express a stably integrated transgene (e.g., 4, 5, 13). We found that transgene expression in Chlamydomonas was significantly improved when the Chlamydomonas HSP70A (A) promoter was fused upstream from the transgene-driving promoter (12,13,14). The A promoter apparently acted as a transcriptional state enhancer; i.e., it improved the probability of randomly integrated transgenes becoming expressed (10,14). Enhancing activities could be assigned to two different regions of the A promoter, a proximal region (ranging from bp Ϫ23 to Ϫ285 upstream from the translational start codon) and a distal region (upstream from bp Ϫ286).The goal of this work was to identify cis-regulatory sequences within the A promoter by means of which it mediates activation of transgene expression. To this end, A promoter deletions and mutations were fused upstream from the RBCS2 promoter (R) to drive expression of the ble gene, conferring resistance to zeocin (9). In earlier work, we estimated the activation efficiency of A promoter derivatives (i) by counting zeocin-resistant colonies produced by cells directly transformed with R-ble/AR-ble constructs and (ii) by determining the fraction of ble-expressing, zeocin-resistant arginine-prototrophic transformants that emerged from arginine-auxotrophic cells cotransformed with the ARG7 gene and R-ble/AR-ble constructs (14). As these methods were tedious and/or led to statistically insignificant results, we sought for an alternative assay to quantify the activating effect of the A promoter. We reasoned that it might be possible to quantify the amount of transgene transcript produced per intact transgene in pools of hundreds of cotransformants generated with the ARG7 gene and R-ble/AR-ble constructs. As in our hands around 20 to 60% of cotransformants contain the cotransformed construct and in case of R-ble ϳ20% of the transgenes are expressed (14), the R-ble construct was expected to be expressed in only 4 to 12% of the cotransformants. To test whether Northern analysis was sensitive enough to detect such low ble mRNA levels in pools of cotransformants, a liquid culture in TAP medium (6) of a Chlamydomonas strain containing an expressing AR-ble construct was increasingly diluted with a culture of a strain containing a nonexpressing R-ble construct (all transformants were generated with strain cw15-302, kindly provided by R. Matagne, University of Liège, Belgium). Northern analysis of RNA extracted from these cells (as described in reference 7) revealed that ble transcript was detectable when only 1% of the cells expres...