Expression of small heat shock protein (sHSP) genes in the garden pea (Pisum sativum) under slow horizontal clinorotation

Abstract

Plant cells respond to stress conditions, such as high temperatures, by synthesizing small heat shock proteins (sHSPs). sHSPs are molecular chaperones that assist in protein folding and prevent irreversible protein aggregation. Although many sHSP genes are temperature-inducible, other variables, such as altered gravity, can induce significant changes in plant cell gene expression. Furthermore, not all subfamilies of sHSP genes share the same expression pattern. The objective of our research was to determine the effect of simulated microgravity (clinorotation) on the expression of sHSP gene subfamilies with different subcellular locations in etiolated pea (Pisum sativum) seedlings. sHSP gene expression levels were examined using quantitative real-time RT-PCR (qPCR). qPCR results demonstrated that sHSP genes were constitutively expressed in seedlings. High temperatures increased the expression of sHSP genes by several thousand-fold. However, simulated microgravity did not have any significant effects on sHSP gene expression.

Introduction

One of the key areas of research in modern space biology concerns the molecular mechanism of plant cell adaptation to microgravity. Studies examining the effects of microgravity on plants during spaceflight have demonstrated the following: higher plants can grow and develop in spaceflight; microgravity does not significantly alter plant morphogenesis, cytogenesis or cell differentiation; and plant cells are gravity-sensitive, as evidenced by cytoskeletal rearrangements and by changes in the balance of intracellular calcium ions.^1,2

The development of modern methods such as microarrays and 2-D gel electrophoresis has enabled the study of the molecular basis of plant cellular responses to spaceflight. Studies have shown that microgravity^3-6 and its partial simulation by random positioning machines^7-9 and clinostats¹⁰ in ground-based experiments, alters the function and transcriptional activity of multiple genes. Studies indicate that there are a number of genes that display moderate but highly significant changes in expression during spaceflight in Arabidopsis (Col-0) seedlings,^3-5 undifferentiated callus tissue⁵ and single-cell model system of Ceratopteris richardii spores.⁶ Among them are genes encoding proteins involved in pathogen defense and environmental stress responses.^3-6

One of the crucial elements of plant adaptation to unfavorable conditions is the so-called “protein quality control network” of molecular chaperones and proteases. Molecular chaperones are a group of diverse proteins that bind to other proteins that are in unstable conformations, thereby preventing their aggregation.^11,12 Small heat shock proteins (sHSPs) are important components of this system. Higher plants possess 11 subfamilies of sHSPs that are highly conserved in both di- and monocotyledons. Six of these subfamilies function in the cytoplasm and nucleus, while the remainders function in cellular organelles. The organellar subfamilies include one subfamily that functions in the endoplasmic reticulum (ER), another that acts in peroxisomes (Po), one that functions in chloroplasts (Cl) and 2 that function in mitochondria (MTI and MTII). This diversity of sHSP subfamilies is a unique feature of plant cells.

In recent years, our understanding of sHSP gene expression in plant tissues expanded as a result of genome-wide studies of a large number of environmental conditions. sHSPs are expressed in response to external factors, such as high and low temperatures, UV radiation, heavy metals, etc. It is known that sHSPs have complicated expression patterns and are constitutively expressed at certain developmental stages in some tissues and organs. These proteins are expressed during embryogenesis, seed maturation, pollen development and fruit maturation.^13-15 The fact that the expression of many plant heat shock genes is altered by microgravity prompted us to analyze sHSP gene expression under this condition.

In this study, we examined the expression of 5 sHSP genes belonging to subfamilies with different subcellular locations, cytosol/nucleus - Pshsp17.1-СІІ and Pshsp18.1-СІ, cloroplasts - Pshsp26.2-Cl, endoplasmatic reticulum - Pshsp22.7-ER and mitochondria - Pshsp22.9-M, in etiolated pea seedlings grown under simulated microgravity.

Results

Constitutive expression of sHSPs in pea seedlings

The results of a qPCR analysis of sHSP gene expression in etiolated pea seedlings are shown in Figures 1 and 2. sHSP gene transcripts were detected throughout the entire period of seedling growth (days 1–5). The highest expression levels (relative to the 5 d old control plants) for all 5 genes occurred on day 1 and were about 45-fold for cytosolic/nuclear subfamilies. mRNA levels decreased thereafter, reaching minimum levels on days 4 and 5. Among all of the genes, cytosolic/nuclear Pshsp17.1 (Fig. 1, А) exhibited the highest levels of constitutive expression, while Pshsp22.7 had the highest expression among the sHsp genes of cellular organels (Fig. 2, C). Other sHSP genes localized in chloroplasts and mitochondria exhibited lower constitutive expression levels (Fig. 2).

Figure 1. Expression of cytoplasmic/nuclear small heat shock protein genes: C-II subfamily (A) and C-I subfamily (B) in etiolated pea seedlings in stationary control and under clinorotation. Error bars on RT-qPCR results represent standard deviations.

Figure 2. Expression of chloroplasts (A), mitochondrion (B) and endoplasmatic reticulum (C) sHSP genes in etiolated pea seedlings in stationary control and under clinorotation. Error bars on RT-qPCR results represent standard deviations.

Expression of sHSPs under slow clinorotation

The objective of clinorotation is to neutralize the effect of the directional component of the gravitational force vector by having each part of the plant experience a multidirectional gravitational pull, thereby simulating microgravity. sHSP gene expression in 1–5 d-old etiolated peas grown under clinorotation was similar to that of the stationary control (Figs. 1, 2). Expression levels were highest on day 1 and decreased thereafter.

sHSP gene expression profiles under high temperatures

Heat shock expreriments were conducted to determine the effects of temperatue on the expression of sHSP genes. sHSP gene expression analysis of 5 day-old pea seedlings confirmed the extreme sensitivity of these genes to high temperatures (Fig. 3 and 4) and demonstrated that etiolated pea seedling can rapidly acclimate to high temperature. mRNA synthesis of all sHSP genes increased significantly, relative to controls, at 30 °C, and reaches their maximum levels at 42 °C. The most highly induced genes were ER-localized Pshsp22.7, mitochondrial Pshsp22.9 and chloroplast Pshsp26.2. At 42 °C, the expression of these genes increased by up to several thousand-fold relative to control seedlings (Fig. 3). In contrast, the expression levels of cytosolic/nuclear sHSP genes increased by 580-fold maximum for Pshsp17.1 at high temperatures (Fig. 4).

Figure 3. Effects of temperature elevation on chloroplasts (A), mitochondrial (B) and endoplasmatic reticulum (C) sHsp genes expression in etiolated pea seedlings. Error bars on RT-qPCR results represent standard deviations.

Figure 4. Effects of temperature elevation on the expression cytoplasmic/nuclear - C-II subfamily (A) and C-I subfamily (B) - sHSP genes in etiolated pea seedlings. Error bars on RT-qPCR results represent standard deviations.

Discussion

As a group, sHSPs are generally expressed in response to cellular stress. Most sHSP genes are expressed at their highest levels under heat shock and some are only expressed during heat stress. However, not all sHSPs are induced by stress. The chaperone function of sHSPs is often linked to the stage of development¹⁴ and it has been reported that sHSP mRNAs and peptides are constitutively expressed in dry pea seeds and seedlings at early growth stages.^12,16

We demonstrated that mRNA transcripts of sHSP genes are constitutively expressed in pea seedlings during early stages of growth, which confirms previous findings.¹⁶ The sHSP mRNAs and peptides found shortly after seed germination are likely residuals of a larger pool of molecules that accumulate during seed maturation and desiccation.¹⁷ However, our results suggest that de novo expression of sHSP genes may occur in some organs and tissues at early stages of seedling development. Subfamilies of sHSPs differed in their relative levels of expression, with the cytosolic/nuclear subfamily genes Pshsp18.1 and Pshsp17.1 exhibiting the highest constitutive levels of expression.

Heat shock experiments confirmed the temperature sensitivity of sHSP genes. The critical role of organelle-localized sHSPs in the cellular adaptation to heat shock is clearly indicated by their mRNA expression levels, which increased up to several thousand-fold at high temperatures.

This study also examined the effect of microgravity on sHSP transcript accumulation in young seedlings. A number of genome-scale studies of other plant species suggest that sHSPs are a part of the adaptive response to microgravity.^3-6,17 Based on these studies, it was concluded that plant cells respond to microgravity as if they were stressed and that the induction of some heat shock genes is a part of this response.⁵ It was also proposed that the induction of sHSPs in microgravity plays a role in stabilizing the cytoskeleton and promoting cell signaling.¹⁷ It has also been reported that plant cells of different model systems, such as etiolated seedlings and cell cultures, undergo adaptive changes in gene expression during spaceflight, although this occurs through different molecular mechanisms.⁵ Cultured cells respond by overexpressing a set of heat shock proteins, including sHSP genes.^5,17-19 sHSP gene expression under microgravity was also confirmed by studies using simulated microgravity.^8,9

Our results show that slow clinorotation did not significantly affect the expression of sHSP genes belonging to subfamilies that function in different cellular compartments. These results are consistent with other studies that showed that heat shock proteins were not stimulated in thale cress (Arabidopsis thaliana),¹⁷ wheat (Triticum aestívum)¹⁸ and barley (Hordeum vulgare)¹⁹ plants during spaceflight. Based on these studies, it is logical to conclude that simulated microgravity, unlike temperature, does not result in aberrant protein synthesis or folding and, therefore, does not induce changes in sHSP gene expression at the transcriptional level. It should be noted, however, that increases in sHSP gene expression in etiolated seedlings may organ-specific. Thus, our use of whole plants as the source of mRNA may have masked any organ- or cell type-specific increase in sHSP gene expression. Therefore, further analysis of the effects of simulated microgravity on sHSP expression in seedlings should be conducted on individual organs or cell types.

Materials and Methods

Plant materials and growth conditions

Pea seeds were sterilized in 5% sodium hypochlorite for 15 min and washed 2 times with sterile water. Seeds were kept in water for 3 h at room temperature. Afterward, every seed was rolled in moist filtered paper tubes and left for 24 h at 4 °C to synchronize seed germination. Seedlings were grown in tubes under slow horizontal clinorotation (2 rpm) or as stationary controls in darkness at 24–26 °C and 45–64% relative humidity for 5 d. Stationary control plants were grown in vertical positions in the same cultivation box. Paper tubes were moistened with 0.5 ml of water every 24 h. For heat shock experiments, seedlings were subjected to the procedure of Wehmeyer et al.,¹⁶ in which the temperature was increased gradually at a rate of 4 °C h⁻¹ to a maximum of 42 °C. At the end of the experiment, seedlings were immediately frozen in liquid nitrogen and preserved at -80 °C.

Quantitative real-time RT-PCR (qPCR)

Total RNA was isolated from seedlings using the Spectrum™ Plant Total RNA Kit (Sigma, catalog number STRN50–1KT) as described in the manufacturer’s protocol and quantified by spectrophotometry. RNA integrity was verified by agarose gel electrophoresis under denaturing conditions and by spectrophotometric assessment of the 260/280 nm ratio. First strand cDNA synthesis was performed using 1 μg of total RNA pre-treated with DNase I (Thermo Scientific, catalog number EN0525) using oligo-(dT)₁₈ primers and the Maxima H Minus Reverse Transcriptase (Thermo Scientific, catalog number EP0751) according to the manufacturer’s instructions. RT-qPCR reactions were performed by using primers specific for pea sHSPs in a Maxima SYBR Green qPCR Master Mix (Thermo Scientific, catalog: K0241). The actin 3 housekeeping gene was used as an internal control. Primer sequences were designed with NCBI ESTs by using Vector NTI Oligo Design tool (Table 1). Primer specificity and the formation of primer dimers were controlled by melting curve analysis and by agarose gel electrophoresis. Primer concentrations were optimized and amplification efficiency was tested by relative standard curves, while specificity of the RT-qPCR amplification was confirmed by melting curve analysis of each single reaction. The real-time PCR was performed in a 25 µl reaction mixture using an iCycler iQ system (Bio-Rad). The following program was applied: initial denaturation: 95 °C, 10 min; followed by 40 cycles at 94 °C, 20 s; 61 °C, 30 s; 72 °C, 30 s.

Table 1. List of genes and primers used for real-time PCR amplification.

Gene	EST	Primer Sequences
Pshsp 18.1 - CI	M33899.1	5′-ttcaccttcc gcttcattcc ct-3′ 5′-ttctcaacgc ttctctctcc gctt-3′
Pshsp17.7 - CII	M33901.1	5′-ataatggacc tcaccgacga caca-3′ 5′-tcttctctct tcctctcgcc acct-3′
Pshsp 22.7 - ER	M33898.1	5′-gattctccca acactctctt atcgg-3′ 5′-ttcctctcac cactcactct tagcac-3′
Pshsp26.2 - P	X07187.1	5′-gtagaaagaa agcctcgaga ag-3′ 5′cacgaatctc acctcctcca atgt-3′
Pshsp22.9-M	X86222	5′-atgtttatcg tcactcctt mL-3′ 5′-attgtccgtc agtaaatcca-3′
Psactin3	U81046.1	5′-tggctacact ttcaccactt ctgc-3′ 5′-ttcagggcat cggaatcttt cagc-3′

Relative expression was calculated based on qPCR efficiency (E) and the threshold values difference (ΔC_t) of treated vs. control samples for both target and reference genes was calculated according to the mathematical model previously described by Pfaffl et al.²⁰ All calculations used C_t values of 5 day-old seedlings from each experimental group (heat shock, control, clinorotation). No reverse transcriptase and no template controls were included in each qPCR run. All experiments were performed in 2 biological replicates (n ≥ 3) and 2 technical replicates (n ≥ 2).