Cloning and characterization of acid invertase genes in the roots of the metallophyte Kummerowia stipulacea (Maxim.) Makino from two populations: Differential expression under copper stress

https://doi.org/10.1016/j.ecoenv.2014.02.005Get rights and content

Highlights

  • Four acid invertases were cloned from two populations of Kummerowia stipulacea.

  • Variations in the protein sequences of acid invertase have no essence influence.

  • The Cu stress elevates the acid invertase transcript level in root of mined plants.

Abstract

The roots of metallophytes serve as the key interface between plants and heavy metal-contaminated underground environments. It is known that the roots of metallicolous plants show a higher activity of acid invertase enzymes than those of non-metallicolous plants when under copper stress. To test whether the higher activity of acid invertases is the result of increased expression of acid invertase genes or variations in the amino acid sequences between the two population types, we isolated full cDNAs for acid invertases from two populations of Kummerowia stipulacea (from metalliferous and non-metalliferous soils), determined their nucleotide sequences, expressed them in Pichia pastoris, and conducted real-time PCR to determine differences in transcript levels during Cu stress. Heterologous expression of acid invertase cDNAs in P. pastoris indicated that variations in the amino acid sequences of acid invertases between the two populations played no significant role in determining enzyme characteristics. Seedlings of K. stipulacea were exposed to 0.3 µM Cu2+ (control) and 10 µM Cu2+ for 7 days under hydroponics׳ conditions. The transcript levels of acid invertases in metallicolous plants were significantly higher than in non-metallicolous plants when under copper stress. The results suggest that the expression of acid invertase genes in metallicolous plants of K. stipulacea differed from those in non-metallicolous plants under such conditions. In addition, the sugars may play an important role in regulating the transcript level of acid invertase genes and acid invertase genes may also be involved in root/shoot biomass allocation.

Introduction

Sucrose can be hydrolyzed to its components, glucose and fructose, by invertase (EC 3.2.1.26). A variety of invertases have been reported, even from individual plant species. Three invertase isoenzyme groups have been distinguished by their solubility, subcellular location, pH optima, and isoelectic points: vacuolar (Inv-V), cell wall (Inv-CW), and alkaline/neutral (Inv-A/N) invertases (Roitsch and Gonzalez, 2004). Inv-Vs and Inv-CWs are acidic invertases, possessing similar enzymatic and biochemical properties and sharing a high degree of overall sequence homology. The former, soluble acidic invertases, are located in the vacuole; they regulate sucrose levels in the vacuole and the remobilization of sucrose for metabolic processes (Roitsch and Gonzalez, 2004). Inv-CWs, ionically bound to the cell wall, cleave sucrose molecules leaked or transported by an efflux sucrose transporter from the sieve element of the phloem into the apoplast (after which the product hexoses are transported into the sink cells by hexose transporters). In contrast to acidic invertases, Inv-A/Ns are not glycosylated and do not belong to the β-fructofuranosidase family. Inv-A/N activity was shown to be strongly inhibited by its hydrolysis products and is not affected by heavy metals, suggesting a markedly different catalytic site to that of acidic invertases (Vargas and Salerno, 2010). Acidic invertases comprise the majority of sucrose-cleaving enzymes in plant meristematic tissues, such as in roots, where there exists a demand for hexose. Acid invertases appear to be involved in plants׳ responses to abiotic and biotic stress, such as pathogen invasion (Sturm and Tang, 1999), salinity (Balibrea et al., 2003), osmotic stress (Wang et al., 2000), and heat (Li et al., 2012). Maintenance of primary metabolic pathways and carbohydrate balance is fundamental to counteracting stress effectively (Stobrawa and Lorenc-Plucinska, 2007).

Molecular biological studies have been carried out to isolate cDNAs or genes encoding cell wall and vacuolar invertases from several higher plants. Isolation of genomic and cDNA clones for different invertases has made it possible to elucidate molecular mechanisms behind the physiological functions of individual members of the invertase gene family during development and under different environmental stimuli. In recent years, many studies have reported invertase activity changes due to gene mutations, e.g., in tomato (Fridman et al., 2004), maize (Carlson et al., 2000, Chourey et al., 2006), Arabidopsis thaliana (Lammens et al., 2008), and bamboo (Chen et al., 2009). Accordingly, we constructed a study to compare the functional characteristics of acid invertases of two populations through heterologous expression of their cDNAs in Pichia pastoris. This expression system has already been successfully used for expressing fructan biosynthesis and degradation enzymes, as well as for invertases (De Coninck et al., 2005, Huang et al., 2003, Wang et al., 2005).

Our previous investigations revealed significantly higher activities of vacuolar and cell wall invertases in the roots of plants from a copper-tolerant population than in plants from a non-copper-tolerant population in Kummerowia stipulacea (Xiong et al., 2008), Rumex dentatus (Huang et al., 2008), and Rumex japonicus (Huang et al., 2011). Furthermore, positive correlations between the activity of acid invertases and root growth and the root/shoot ratio were observed (Huang et al., 2008, Xiong et al., 2008). In addition, high acid invertase enzyme activities accompanied elevated expression of their genes in seeds (Huang et al., 2011). These studies suggested that the higher activity of acid invertases in heavy metal-tolerant plant populations might play a role in the maintenance of such heavy metal tolerance by supplying carbon and energy for supporting tolerance mechanisms. At the molecular level, such elevated activities in acid invertases may be the result of invertase gene mutation and altered expression. To understand the putative roles of invertase genes in changes in enzyme activity in metallophyte plants, we isolated cell wall invertase and vacuolar invertase genes from the roots of Cu-tolerant and non-tolerant populations of K. stipulacea and expressed their cDNAs in the yeast P. pastoris system. Comparison of the kinetics parameters of these cDNAs would enable determination of whether alterations in the amino acid residues in the translated proteins could influence enzyme characteristics. Finally, differences in expression levels of the acid invertases between the two populations under copper stress were studied.

Section snippets

Plant materials and culture

Two natural populations of K. stipulacea were selected for study, a mine population (MP) growing on an ancient waste heap of a Cu mine, located on Tonglushan Hill, Hubei Province, China, and a non-mine population (NMP) from the campus of Wuhan University, Wuhan, China. These populations represented Cu-tolerant and non-tolerant populations, respectively. In each population, large quantities of seeds were sampled from more than 100 randomly selected plants and pooled. Seeds for the subsequent

Cloning and characterization of acid invertase cDNAs

The entire cDNA sequences of the acid invertase genes of K. stipulacea were assigned to GenBank under the accession numbers JQ041362, JQ041363, JQ041364, and JQ041365 (www.ncbi.nlm.nih.gov/GenBank). In this study, the encoded proteins are referred to as CV-inv (vacuolar invertases from the Cu-tolerant population), NCV-inv (vacuolar invertases from the non-Cu-tolerant population), CCW-inv (cell wall invertases from the Cu-tolerant population), and NCW-inv (cell wall invertases from the

Discussion

In this study, we isolated four acid invertase genes, two cell wall invertases, and two vacuolar invertases, from the cDNA library of young roots of Cu-tolerant and non-tolerant populations of K. stipulacea. The deduced amino acid sequences of the four genes showed high similarity to the acid invertases of other species (Fig. 1), all of which contain the highly conserved β-fructosidase motifs NDPNG/A and catalytic sites WECP/VD (Goetz and Roitsch, 1999). A phylogenetic tree we constructed based

Acknowledgments

The research was supported by the National Nature Science Foundation of China (Projects 20677046, 30870365, and 31270432).

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