HSP90 canonical content organizes a molecular scaffold mechanism to progress flowering.

Highly interactive signaling processes constitute a set of parameters intertwining in a continuum mode to shape body formation and development. A sophisticated gene network is required to integrate environmental and endogenous cues in order to modulate flowering. However, the molecular mechanisms that coordinate the circuitries of flowering genes remain unclear. Here using complemented experimental approaches, we uncover the decisive and essential role of HEAT SHOCK PROTEIN 90 (HSP90) in restraining developmental noise to an acceptable limit. Localized depletion of HSP90 mRNAs in the shoot apex resulted in low penetrance of vegetative-to-reproductive phase transition and completely abolished flower formation. Extreme variation in expression of flowering genes was also observed in HSP90 mRNA-depleted transformed plants. Transient heat-shock treatments moderately increased HSP90 mRNA levels and rescued flower arrest. The offspring had a low, nevertheless noticeable failure to promote transition from vegetative into the reproductive phase and showed flower morphological heterogeneity. In floral tissues a moderate variation in HSP90 transcript levels and in the expression of flowering genes was detected. Key flowering proteins comprised clientele of the molecular chaperone demonstrating that the HSP90 is essential during vegetative-to-reproductive phase transition and flower development. Our results uncover that HSP90 consolidates a molecular scaffold able to arrange and organize flowering gene network and protein circuitry, and effectively counterbalance the extent to which developmental noise perturbs phenotypic traits.

Brassinosteroid nuclear signaling recruits HSP90 activity.

Heat shock protein 90 (HSP90) controls a number of developmental circuits, and serves a sophisticated and highly regulatory function in signaling pathways. Brassinosteroids (BRs) control many aspects of plant development. Genetic, physiological, cytological, gene expression, live cell imaging, and pharmacological approaches provide conclusive evidence for HSP90 involvement in Arabidopsis thalianaBR signaling. Nuclear-localized HSP90s translocate to cytoplasm when their activity is blocked by the HSP90 inhibitor geldanamycin (GDA). GDA treatment promoted the export of BIN2, a regulator of BR signaling, from the nucleus into the cytoplasm, indicating that active HSP90 is required to sustain BIN2 in the nucleus. HSP90 nuclear localization was inhibited by brassinolide (BL). HSP90s interact with BIN2 in the nucleus of untreated cells and in the cytoplasm of BL-treated cells, showing that the site-specific action of HSP90 on BIN2 is controlled by BRs. GDA and BL treatments change the expression of a common set of previously identified BR-responsive genes. This highlights the effect of active HSP90s on the regulation of BR-responsive genes. Our observations reveal that HSP90s have a central role in sustaining BIN2 nuclear function. We propose that BR signaling is mediated by HSP90 activity and via trafficking of BIN2-HSP90 complexes into the cytoplasm.

Novel aspects of transcriptional regulation in the winter survival and maintenance mechanism of poplar.

Temperate woody plants have developed sophisticated winter survival and maintenance mechanisms that enable them to adapt rapidly to the annual cycle of environmental changes. Here, we demonstrate notable aspects of the transcriptional regulation adopted by poplar in winter/dormancy, employing biochemical and whole transcriptome analysis, and showing high levels of transcriptional activity in a broad spectrum of genes during the dormancy period. A total of 3237 probe sets upregulated more than threefold in winter/dormancy stems over summer/active-growth stems were identified. As expected, genes related to cold hardiness and defense were over-represented. Carbohydrate biosynthesis and transport-related genes were also actively expressed in winter/dormancy stems. Further biochemical analyses verified the dormancy/winter transcription phenotype. More than 60% of the winter upregulated transcription factors (TFs) were related to either biotic or abiotic stress. This finding substantiates that the major transcriptional network of winter/dormancy stems is related to stress tolerance, such as dehydration, cold tolerance and defense. Furthermore, during winter/dormancy, preferential expression of genes involved in cell wall biosynthesis or modification, indirect transcriptional regulation (RNA metabolism) and chromatin modification/remodeling were observed. Taken together, these findings show that regulation of gene expression associated with winter survival and maintenance extends beyond control by promoter-binding TFs to include regulation at the post-transcriptional and chromatin levels.

Subtle proteome differences identified between post-dormant vegetative and floral peach buds.

Proper development of deciduous tree species, including peach, is accomplished through an annual growth cycle. Freezing avoidance during winter is necessary for tree survival and is achieved by the enclosure of meristems in floral and vegetative buds. To elucidate the role of developmentally regulated protein networks in bud break, proteins of the two bud-types were extracted and analyzed by two-dimensional gel electrophoresis (2-DE). Of the 1107 protein spots that were picked, 475 were identified and annotated assembling the peach bud proteome reference map. The majority of these proteins are involved in stress-response, detoxification, defense, carbohydrate metabolism and energy production. The protein profiles of both bud-types bear high similarity, whereas only 11 proteins were differentially expressed. These proteins were mainly involved in carbon-nitrogen homeostasis/metabolism and certain developmental processes to sustain rapid growth of the newly emerging organs. Among these are enzymes that differentially regulate the levels of H(2)O(2) between floral and vegetative buds, potentially promoting sequential bud-break. Distinct Nucleoside Diphosphate Kinase (NDPK) variants in floral and vegetative buds were detected suggesting the potential role of NDPKs in H(2)O(2)-mediated signaling for post-dormant bud break. This study provides data towards a better understanding of dormancy release and bud break.

Rootstock-induced dwarfing in cherries is caused by differential cessation of terminal meristem growth and is triggered by rootstock-specific gene regulation.

Use of dwarfing rootstocks has dramatically increased the profitability of fruit production by reducing production costs, reduced chemical use and higher density plantings. Despite the importance of rootstock-induced dwarfing, the cause of this phenomenon is not known. Using two commercially available graft combinations consisting of a sweet cherry scion, 'Bing', on a dwarfing rootstock (Gi5) or a semi-vigorous rootstock (Gi6), we discovered that the difference in grafted tree height was due to a significantly earlier cessation of terminal meristem growth of the scion on Gi5 compared to Gi6 rootstock, rather than shorter metamer length. We then carried out cDNA-AFLP analysis to investigate differential gene expression between the two graft combinations. Transcript-derived fragments (TDFs) identified as differentially expressed were cloned and printed on microarrays for further confirmation of the differential expression. A total of 99 TDFs were identified as differentially expressed between the 'Bing'/Gi5 and 'Bing'/Gi6 samples, including genes involved in transcription regulation, brassinosteroid signaling, flavonoid metabolism and cell wall biosynthesis or modification. Rootstock vigor has a significant effect on gene expression at the scion and the graft union. Timing of the differential gene expression in the dwarf trees coincides with the earlier cessation of terminal shoot growth, suggesting that these differentially expressed genes may be involved in the dwarfing phenomenon.

Complexity of Hsp90 in organelle targeting.

Heat shock protein 90 (Hsp90) is an abundant and highly conserved molecular chaperone. In Arabidopsis, the Hsp90 gene family consists of seven members. Here, we report that the AtHsp90-6 gene gives rise to two mRNA populations, termed AtHsp90-6L and AtHsp90-6S due to alternative initiation of transcription. The AtHsp90-6L and AtHsp90-6S transcription start sites are located 228 nucleotides upstream and 124 nucleotides downstream of the annotated translation start site, respectively. Both transcripts are detected under normal or heat-shock conditions. The inducibility of AtHsp90-6 mRNAs by heat shock implies a potential role of both isoforms in stress management. Stable transformation experiments with fusion constructs between the N-terminal part of each AtHsp90-6 isoform and green fluorescent protein indicated import of both fusion proteins into mitochondria. In planta investigation confirmed that fusion of the AtHsp90-5 N-terminus to green fluorescent protein (GFP) did result in specific chloroplastic localization. The mechanisms of regulation for mitochondria- and plastid-localized chaperone-encoding genes are not well understood. Future work is needed to address the possible roles of harsh environmental conditions and developmental processes on fine-tuning and compartmentalization of the AtHsp90-6L, AtHsp90-6S, and AtHsp90-5 proteins in Arabidopsis.

Developmental and seasonal expression of PtaHB1, a Populus gene encoding a class III HD-Zip protein, is closely associated with secondary growth and inversely correlated with the level of microRNA (miR166).

In contrast to our knowledge of the shoot apical meristem, our understanding of cambium meristem differentiation and maintenance is limited. Class III homeodomain leucine-zipper (HD-Zip) proteins have been shown to play a regulatory role in vascular differentiation. The hybrid aspen (Populus tremulaxPopulus alba) class III HD-Zip transcription factor (PtaHB1) and microRNA 166 (Pta-miR166) family were cloned from hybrid aspen using a combination of in silico and polymerase chain reaction methods. Expression analyses of PtaHB1 and Pta-miR166 were performed by Northern blot analysis. The expression of PtaHB1 was closely associated with wood formation and regulated both developmentally and seasonally, with the highest expression during the active growing season. Also, its expression was inversely correlated with the level of Pta-miR166. Pta-miR166-directed cleavage of PtaHB1 in vivo was confirmed using modified 5'-rapid amplification of cDNA ends (RACE). The expression of Pta-miR166 was much higher in the winter than in the growing seasons, suggesting seasonal and developmental regulation of microRNA in this perennial plant species.

Transcriptome profiling of vertical stem segments provides insights into the genetic regulation of secondary growth in hybrid aspen trees.

In order to better understand the genetic regulation of secondary growth in hybrid aspen (Populus tremula L.xP. alba L.), we carried out a series of cDNA-amplified fragment length polymorphism (AFLP)-based transcriptome analyses in vertical stem segments that represent a gradient of developmental stages with regard to secondary growth. This approach allowed us to screen >80% of the transcriptome expressed in six samples and identify genes differentially expressed with the progress of secondary growth, in a tissue-specific manner. Of the 76,800 transcript-derived fragments (TDFs) analyzed, 271 TDFs were selected and sequenced based on their differential expression patterns. Many of the xylem-up-regulated genes were involved in cell wall and lignin biosynthesis, while the bark-up-regulated genes had diverse functional roles. About 25% of the xylem-up-regulated TDFs analyzed were involved in the phenylpropanoid biosynthesis pathway, which produces the cell wall polymer lignin and various wood extractives. In addition, many of the TDFs showing secondary xylem-specific expression were annotated as genes not previously reported in Populus, including novel cell death proteins, cytoskeleton-interacting proteins, transporters and putative transcription factors.