The plant sHSP superfamily: five new members in Arabidopsis thaliana with unexpected properties

The small heat shock proteins (sHsps), which are ubiquitous stress proteins proposed to act as chaperones, are encoded by an unusually complex gene family in plants. Plant sHsps are classified into different subfamilies according to amino acid sequence similarity and localization to distinct subcellular compartments. In the whole Arabidopsis thaliana genome, 19 genes were annotated to encode sHsps, of which 14 belong to previously defined plant sHsp families. In this paper, we report studies of the five additional sHsp genes in A. thaliana, which can now be shown to represent evolutionarily distinct sHsp subfamilies also found in other plant species. While two of these five sHsps show expression patterns typical of the other 14 genes, three have unusual tissue specific and developmental profiles and do not respond to heat induction. Analysis of intracellular targeting indicates that one sHsp represents a new class of mitochondrion-targeted sHsps, while the others are cytosolic/nuclear, some of which may cooperate with other sHsps in formation of heat stress granules. Three of the five new proteins were purified and tested for chaperone activity in vitro. Altogether, these studies complete our basic understanding of the sHsp chaperone family in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel small heat shock protein of <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>: characterization,structure modeling,and expression profiles under environmental stresses

Small heat shock proteins (sHsps) are a class of chaperones with low molecular weight, feathered by a C-terminal α-crystallin domain (ACD). They participate in reestablishing the stability of partially denatured proteins and therefore contribute to cellular homeostasis. In this work, we identified a sHsp homolog (designated as sHsp19) from Haliotis discus hannai, an economically important farmed mollusk in East Asia. sHsp19 possesses a sHsp hallmark domain, which exhibits the typical fold of ACD as revealed by a three-dimensional model constructed through an iterative threading assembly refinement method. The amino acid sequence sHsp19 shares low identities with any other known sHsps, with percentages below 35 %. Besides, sHsp19 shows relatively distant phylogenetic relationships with sHsps of various mollusks, including two other identified sHsps of abalone subspecies. qRT-PCR analysis indicated that the expression of sHsp19 occurred in multiple tissues. Upon exposure to thermal, oxidative, and multiple toxic metal stresses, the level of sHsp19 mRNA was rapidly elevated in a persistent fashion, with the maximum increase up to 170.58-, 405.84-, and 361.96-fold, respectively. These results indicate sHsp is a novel sHsp that possesses the distinguishing structural feature of sHsps but has remote homologies with known sHsps. It is likely to be important in stress adaptation of abalone and may be applied as a bioindicator for monitoring pollution or detrimental changes of environment in abalone culture.     

Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation.

The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers.     

Small heat shock proteins are differentially regulated during pollen development and following heat stress in tobacco

In plants small heat shock proteins (sHsp) are abundantly expressed upon heat stress in vegetative tissue, however, sHsp expression is also developmentally induced in pollen. The developmental induction of sHsp has been related to the potential for stress-induced microspore embryogenesis. We investigated the polymorphism among sHsp and their expression during pollen development and after heat stress in tobacco. Real-time RT-PCR was used for quantification of mRNA of two known and nine newly isolated cDNAs representing cytosolic sHsp. At normal temperature most of these genes are not transcribed in vegetative tissues, however, all genes were expressed during pollen development. Low levels of mRNAs were found for sHsp-1A and -1B in early-unicellular stage, increasing four to sevenfold in mature pollen. Nine other genes are up-regulated in unicellular and down-regulated in bicellular pollen; three these genes show stage-specific expression. Western analysis revealed that cytosolic class I and II sHsp are developmentally expressed during all stages of pollen development. Different subsets of cytosolic sHsp genes are expressed in a stage-specific fashion suggesting that certain sHsp genes may play specific roles in early, others during later stages of pollen development. Heat stress results in a relatively weak and incomplete response in pollen: (i) the heat-induced levels of mRNA (excepting sHsp-2B, −3Cand -6) are much lower than in leaves, (ii) several sHsp are not detected after heat stress in pollen, although, they are heat-inducibly expressed in leaves. Application of heat stress, cold, and starvation, which induce microspore embryogenesis, modify mRNA levels and the patterns of 2-D-separated sHsp, but only heat stress enhances the expression of sHsp in microspores. There is no correlation of the expression of specific sHsp with the potential for microspore embryogenesis.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Pollen Embryogenesis - The Stress Mediated Switch from Gametophytic to Sporophytic Development. Current Status and Future Prospects

Embryogenesis can be initiated directly from microspores or pollen grains. This is known as androgenesis and refers to the process of redirection of normal pollen development (gametophytic pathway) towards the embryo formation (sporophytic). This review mainly deals with the current knowledge of stress and developmental aspects of induction of androgenesis. The crucial role of stress inductive treatment together with changes in cell polarity are discussed in relation to other relevant biological systems. The intriguing speculations are made on the basis of these comparisons which may point out the direction of future investigations.     

The identification and characterization of IbpA,a novel α-crystallin-type heat shock protein from mycoplasma

α-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present in Acholeplasma laidlawii. Importantly, similar to the endogenously expressed sHsp proteins, the recombinant IbpA protein was able to spontaneously generate oligomers in vitro and to rescue chemically denatured bovine insulin from irreversible denaturation and aggregation. Collectively, these data suggest that IbpA is a bona fide member of the sHsps family. The immune-electron microscopy data using specific antibodies against IbpA have revealed different intracellular localization of this protein in A. laidlawii cells upon heat shock, which suggests that IbpA not only may participate in the stabilization of individual polypeptides, but may also play a protective role in the maintenance of various cellular structures upon temperature stress.     

Proteomic and biochemical analysis of maize anthers after cold pretreatment and induction of androgenesis reveals an important role of anti-oxidative enzymes

In stress conditions, microspores and young pollen grains can be switched from their normal pollen development toward an embryogenic pathway via a process called androgenesis. Androgenic embryos can produce completely homozygous, haploid or double-haploid plants. This study aimed to investigate changes in the abundance of protein species during cold pretreatment and subsequent cultivation of maize anthers on induction media using gel-based proteomics. Proteins upregulated on the third day of anther induction were identified and discussed here. Simultaneous microscopic observations revealed that the first division occurred in microspores within this period. Using 2-D electrophoresis combined with MALDI TOF/TOF MS/MS analysis 19 unique proteins were identified and classified into 8 functional groups. Proteins closely associated with metabolism, protein synthesis and cell structure were the most abundant ones. Importantly, ascorbate peroxidase, an enzyme decomposing hydrogen peroxide, was also upregulated. Isozyme analysis of peroxidases validated the proteomic data and showed increased peroxidase activities during androgenic induction. Further, the isozyme pattern of SOD revealed increased activity of the MnSOD, which could provide hydrogen peroxide as a substrate for in vivo peroxidase reactions (including ascorbate peroxidase). Together, these data reveal the role of enzymes controlling oxidative stress during induction of maize androgenesis.     

The chloroplast‐localized small heat shock protein Hsp21 associates with the thylakoid membranes in heat‐stressed plants

The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity of sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps with a single conserved β‐sandwich fold domain defining the family, the α‐crystallin domain, whereas the N‐terminal and C‐terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non‐metazoan sHsp expressed in the chloroplasts in green plants which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat‐stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic ¹⁵N‐isotope labeling of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat‐stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered.     

Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding

Small heat shock proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded proteins, which facilitates subsequent substrate solubilization and refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp–substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp–substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards refolding pathway. The sHsp/Hsp70 interplay has major impact on protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient refolding of damaged proteins under favourable folding conditions.