Elsevier

Process Biochemistry

Volume 35, Issue 6, January 2000, Pages 603-613
Process Biochemistry

A model for enhanced pea seedling vigour following low pH and salicylic acid treatments

https://doi.org/10.1016/S0032-9592(99)00111-9Get rights and content

Abstract

In the simplest of terms, seed vigour is a visual measure of a seed’s ability to germinate and survive its early growth and development period. Improvement of seed vigour is important for optimal emergence, stress resistance and uniform growth of emerging seedlings. We have hypothesized that acid-induced cell growth and elongation is regulated through the pentose-phosphate pathway; therefore, the effect of acidification linked to salicylic acid (SA) on growth, cell elongation, and phenolic synthesis was investigated. The experiments consisted of low pH and SA treatments followed by the measurement of phenolic levels and assay of the key regulatory enzyme of the pentose-phosphate pathway, glucose-6-phosphate dehydrogenase (G6PDH), and guaiacol peroxidase (GPX), during post-germination growth and elongation of peas. Phenolic and enzyme levels were determined by UV spectrophotometric assays. A low pH environment stimulated phenolic synthesis and increased tissue rigidity. Stimulating phenolic synthesis through low pH treatment supports the hypothesis that acid-induced cell growth and elongation may be regulated through the pentose-phosphate pathway. Based on concomitant stimulation of G6PDH and increase in proline content, the pentose-phosphate pathway may be linked to stimulation of proline metabolism in response to the above treatments. It has been hypothesized that this pathway produces the critical precursors for the synthesis of phenolic secondary metabolites that are important for plant growth and lignification.

Introduction

Seed vigour is a term encompassing the sum total of those properties of the seed that determine the potential performance of the seed or seed lot during germination and seedling emergence [1]. Rapid and uniform germination are among the properties of vigourous seeds [2]. Improvement of seed vigour is important for optimal emergence, stress resistance, and uniform growth of emerging seedlings. Seed priming or osmoconditioning are terms to describe a pre-sowing hydration treatment developed to improve seedling establishment [3]. Seed priming or other prehydration treatments can accelerate the rate of germination of many species of seeds and often increase uniformity as well [4]. Seedling emergence of primed seeds is earlier and the emergence is more synchronous, especially when sown under suboptimal temperatures [2].

Improvement or modification of plant growth and development can occur by the direct application of nutrients or plant growth regulators to seeds [3]. The effect of low pH on seed vigour was investigated because proton pumping across the plasma membrane and against a pH gradient by proton-ATPase (H+-ATPase) activity is essential for nutrient uptake, turgor generation, external acidification (Fig. 1a) for cell wall loosening, and cytoplasmic pH regulation [5]. A decrease in intracellular pH has been reported during the hypersensitive reaction of plants to pathogens and may enhance conversion of hydrogen peroxide to the active hydroxyl radicals [6], [7]. Exogenously applied salicylic acid (SA) results in the activation of a range of plant defence genes [8]. The SA level in plants increases in response to infection, exposure to ultraviolet (UV) light and ozone and is believed to be part of a signaling process that results in systemic acquired resistance (SAR) [9], [10], [11]. SA may sensitize cells for rapid defence gene activation by acting as a signal in a transduction pathway that activates defence response genes [12]. Hydrogen peroxide has been implicated as a key component in SAR signaling upstream of salicylate by identifying a peroxidase that is inhibited by the binding of salicylate and SAR-inducing derivatives [13].

Hydrogen peroxide (H2O2) is produced in response to a requirement for peroxidase-mediated cross-linking reactions in the cell wall, where peroxidase substrates, such as hydroxyproline-rich glycoproteins (HRGPs) and phenolic compounds, accumulate [14]. Peroxidase is a hemoprotein that catalyzes the oxidation of many substrates, using hydrogen peroxide as an electron acceptor [15]. In the presence of hydrogen peroxide, peroxidase is thought to mediate the cross-linking between numerous other cell wall components [16]. Polymerization of hydroxycinnamyl alcohols in the final stages of lignin biosynthesis and the intramolecular and intermolecular cross-linking of HRGPs are both catalyzed by isoperoxidase (multiple forms of peroxidase that differ from one another in one or more properties) activities [7].

Each peroxidase isoenzyme group plays a different physiological function in plant cell metabolism [17]. It has been suggested that these enzymes play an important role in several physiological processes including lignin synthesis, indole-3-acetic acid (IAA) metabolism, pathogen resistance, and response to stress [18], [19], [20], [21], [22]. Guaiacol peroxidases (GPXs), e.g. horseradish peroxidase, have a very broad specificity and appear to be involved in a number of physiological processes including the biosynthesis of lignin and ethylene and in the degradation of IAA [23]. In nature, lignin, a complex polymer consisting of phenylpropanoid units interconnected by a variety of carbon–carbon bonds and ether linkages, physically encrusts cellulose and is resistant to degradation by most microorganisms [24], [25]. Enhanced peroxidase activity is believed to lead to lignin biosynthesis, and the subsequent lignification of the cell wall acts as a barrier to fungal invasion [26], [27]. Ascorbate peroxidases are believed to metabolize hydrogen peroxide by using ascorbate as a co-substrate through an ascorbate-dependent pathway [28]. Peroxidases can also generate hydrogen peroxide via hydrogen peroxide-independent oxidation of a number of substrates, for example, NADH, NADPH2, thiols, and certain phenols [29], [30], [31], [32].

Oxidative stress is likely to result whenever environmental conditions block the normal dissipation of the light-induced high-energy state [33]. The reduction in the rate of CO2 assimilation by oxidative stress results in the exposure of chloroplasts to excess excitation energy and increases the rate of formation of reactive oxygen intermediates (free radicals) [34]. In the cell wall, plant phenolics form barriers to prevent moisture loss or diffusion and pathogen encroachment [35]. Plant phenolics have the potential to function as antioxidants by trapping free radicals generated during oxidative processes which then normally undergo coupling reactions leading eventually to polymeric or oligomeric products [35]. Lignins are the most abundant of the phenylpropanoids and are one of the most common classes of plant phenolics having antioxidant properties [35].

In this study, SA, which originates from the phenylpropanoid pathway, has been observed to stimulate phenolic accumulation in peas during and/or following germination and may indirectly stimulate the rate of peroxidase-induced polymerization [36]. Concurrent with phenolic stimulation we investigated whether or not the pentose-phosphate pathway is stimulated by measuring the activity of the first committed step (G6PDH). This stimulation is essential for the synthesis of sugar phosphates and NADPH2 for all anabolic pathways, including auxin, cytokinin, and phenolic synthesis (Fig. 1b).

Shetty (1997) has hypothesized that the same metabolic flux from the induction of the proline-linked pentose-phosphate pathway regulates the conversion of ribose-5-phosphate to erythrose-4-phosphate and drives the shikimate pathway (Fig. 1b), critical for both auxin and phenylpropanoid biosynthesis [37]. A proline-linked PPP regulated by the availability of NADPH2 may help drive the metabolic flux towards the shikimate pathway and related metabolite biosynthesis [37].

In this study the effect of low pH and SA treatments on pea (Pisum sativum) vigour response was quantified by monitoring changes in G6PDH and GPX activities and changes in average height, weight, and total phenolic synthesis during growth and elongation of peas following germination. We also investigated the potential role of acidification linked to SA on growth, cell elongation, and phenolic enhancement.

Section snippets

Plant materials

Whole green pea seeds, P. sativum, (Goya® Foods Inc., Secaucus, NJ) were used for this study.

Seed treatments

Thirty seeds were allowed to imbibe with shaking for 24 h in 50 ml of distilled water (dH2O), 50 μM SA, 100 μM SA, or Trichoderma harizianum extract (TH), a seed vigour enhancer [38], adjusted as needed to the required pH using 0.01 N HCl or 0.01 N NaOH. For the UV experiment, pretreated pea seeds were exposed to UV light (General Electric Corporation, 48”-30W Germicidal UV light, model no. G30T8) for

Results and discussion

Pea seeds (P. sativum) primed with distilled water or SA at pH 3.0 germinated into plants with more biomass and height after 10 days than pea plants from seeds primed with normal-pH distilled water (Table 1). While net proton release by H+-ATPase is essential for nutrient uptake, the low pH treatments may have induced a transitory increased H+ concentration in the intracellular environment in these pea seedlings that enhanced the metabolism for mobilization of nutrient reserves from the

Acknowledgements

We would like to express our thanks to Brian and Nancy McCue for technical assistance and to Jenna Hwang for critical reading of the manuscript and helpful discussions.

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