In vivo role of Arabidopsis arginase in arginine metabolism and abiotic stress response

Abstract

Nitric oxide (NO) and polyamines play essential roles in many developmental processes and abiotic stress responses in plants. NO and polyamines are metabolized from arginine through NO synthase (NOS) and arginine decarboxylase (ADC), respectively. Function of arginase, another important enzyme involved in arginine metabolism, in abiotic stress remains largely unknown. In the recent study, we have dissected the impact of arginase on arginine metabolism and abiotic stress responses through manipulating AtARGAHs expression. The results suggested that manipulation of arginase expression modulated accumulation of arginine and direct downstream products of arginine catabolism. AtARGAHs knockout lines exhibited increased accumulation of polyamines and NO and enhanced abiotic stress tolerance, while AtARGAHs overexpressing lines displayed the opposite results. Notably, we highlighted that Arabidopsis arginase plays distinctive and dual roles in the crosstalk between polyamines and NO signaling during abiotic stress responses, mediating both arginine metabolism and reactive oxygen species (ROS) accumulation. It is likely that accumulation of both NO and polyamines might activate abiotic stress responses in the plant.

Arginine is an important amino acid in plants, as a medium for the transport and storage of nitrogen (N) and a precursor for the synthesis of polyamines and nitric oxide (NO).^1-5 As shown in Figure 1, arginase (also known as ARGAH in Arabidopsis) is responsible for catalyze of arginine to ornithine and urea, and arginine decarboxylase (ADC) can metabolize arginine to agmatine and further to polyamines including putrescine, spermidine, spermine, etc.^2,3,5 Additionally, although nitric oxide synthase (NOS) has not been cloned in higher plants, similar NOS activity of animals has been detected in many plant species by many research groups.^6-11 And it has been confirmed that NO might be generated primarily during the conversion of arginine to citrulline by the NOS, especially in responses to several biotic and abiotic stresses.^6-11

Figure 1. A simple model of arginine metabolism in Arabidopsis. Three enzymes are responsible for arginine metabolism: (1) via arginine to ornithine and urea by arginase; (2) via arginine to NO and citrulline by NOS; (3) via arginine to agmatine by ADC, from agmatine to putrescine by AIH and NLP, and putrescine may be further metabolized to spermidine and spermine. ADC, arginine decarboxylase; AIH, agmatine iminohdrolase; dcSAM, decarboxylated S-adenosyl-l-methionine; NLP, N-carbamoylputrescine amidohydrolase; NO, nitric oxide; NOS, nitric oxide synthase; SPDS, spermidine synthase; SPMS, spermine synthase.

Previous research indicated that knockout of Arabidopsis arginase gene resulted in increased NO accumulation,² and overexpression of AtARGAH2 conferred increased tolerance to Botrytis cinerea and enhanced callus development during clubroot infection.^3,12 Recently, we investigated the influence of manipulation of AtARGAHs expression on arginine metabolism, as well as plant responses to abiotic stresses including water deficit, salt and freezing.⁵

Effect of arginase expression on arginine metabolism

As shown in Figure 1, the balance of arginine level in higher plants are regulated by enzymes directly or indirectly involved in arginine biosynthesis, as well as those enzymes (arginase, ADC and NOS) involved in the down-stream of arginine metabolism.^2-5 If arginase expression was modulated, the direct insight was to examine the arginine accumulation. Although changes of AtARGAHs expression had no significant effect on upstream arginine concentration per g fresh weight (FW), AtARGAHs knockout and overexpressing lines showed significantly less relative arginine (% of total free amino acids) than that of wild type.⁵ All these results were consistent with what Flores et al. (2008) and Brauc et al. (2012) reported.^2,3 It is reasonable that all AtARGAHs overexpressing lines showed decreased relative arginine, indicating a direct effect of AtARGAHs on arginine pools.^2,3,5 Surprisingly, AtARGAHs knockout lines also exhibited decreased relative arginine. NOS like enzyme activity, generating NO directly from arginine, has been examined in chloroplast, peroxisome and mitochondria in plant.^2,5,8 ADC, the first enzyme responsible for polyamine synthesis, is localized in the chloroplast.^3,4 Since it is difficult to examine and quantify chloroplastic, peroxisomal and mitochondrial arginine pools in plant tissues, we can only speculate that the specifically localized arginine concentrations might be affected and metabolized to polyamines and NO.^2,5

Besides arginine pools, we also found that manipulation of arginase expression modulates the down-stream arginine metabolism, including both ADC and NOS pathway. For ADC pathway, the mRNA levels of several major genes (ADC1, ADC2, AIH and NLP1) were significantly influenced, and the metabolism products (putrescine and spermine) were also modulated.⁵ Consistently, overexpression of ADC increased polyamine concentrations in many plant species, including rice, tobacco, Arabidopsis and tomato, etc.^4,5 Additionally, the NOS activity and NO concentration were also modulated through manipulating of arginase expression.⁵

Involvement of Arabidopsis Arginase in Response to Abiotic Stress

NO and polyamines are essential endogenous signaling molecules involved in multiple physiological processes and various stress responses.^6-11,13-24 Previous research has shown that exogenous application of polyamines could induce a rapid NO production.¹ Recently, it was found that application of nitric oxide donor (sodium nitroprusside) could modulate almost all polyamine metabolism genes’ expression (ADC, SAMDC, SPMS, SPDS, etc) and enzyme activities, thus regulated the concentrations of polyamines in Medicago truncatula.¹⁷ Some stress factors might share the activation of stress responsive pathway in the plant, characterized by the production of nitrogen species and reactive oxygen species (ROS).^6-11,13-24 Among multiple signaling molecules, NO and polyamines might be ideal candidates to integrate several environment stress responses, but the crosstalk of NO and polyamines is complex and needs to be further dissected.^1,4,17 As shown in Figure 1, manipulating the expression of AtARGAHs supplied a good version to investigate the link between NO and polyamines, especially in plant responses to abiotic stress.^2-5

Our previous research together with other published data confirmed that proper levels of NO might be necessary and beneficial for plants in response to both abiotic stress and disease resistance.^6-11,13,14 As to polyamines, overexpression of several polyamine metabolism genes including ADC and SPMS resulted in increased polyamines and enhanced abiotic stress tolerance, while knock-down of these genes had the opposite results.^16-23 In addition, exogenous polyamines treatment could abrogate the decreased abiotic stress tolerance in the polyamine-deficent mutant plants.^16-23 Consistently, we found that the knockout mutants and overexpression transgenic plants showed opposite abiotic stress responses and manipulation of arginase expression modulate both ADC and NOS pathway.⁵ Interestingly, AtARGAHs could modulate ROS accumulation and antioxidant enzyme activities under abiotic stress conditions, which is consistent with the results that both polyamines and NO play complex roles to deal with ROS derived from environmental stresses.^4,5,16,21 All these results may give new insights and evidences about crosstalk of NO and polyamines under abiotic stress conditions, and functional link between NO and polyamines metabolisms and ROS accumulation in vivo.^4,5,16,21 We hypothesized that NO and polyamines might share the same pathways to activate rapid stress responses in the plant. However, the underlying molecular and cellular mechanisms of crosstalk between NO and polyamine during abiotic stress responses remain elusive.^4,5 Further studies combining genetic biology and molecular biology techniques will provide more clues to the regulation mechanism of NO and polyamines signaling pathway during plant abiotic stress responses.

Conclusions

Taken together, our experiments highlighted the effect of Arabidopsis arginase expression on arginine metabolism, as well as in plant responses to abiotic stress. The underlying mechanisms of stress responses were also discussed, partly attributed to the in vivo roles of AtARGAHs in the regulation of arginine metabolism and ROS accumulation.

Glossary

Abbreviations:

AIH

agmatine iminohdrolase

ARGAH

arginase

ADC

arginine decarboxylase

dcSAM

decarboxylated S-adenosyl-L-methionine

FW

fresh weight

N

nitrogen

NLP

N-carbamoylputrescine amidohydrolase

NO

nitric oxide

NOS

nitric oxide synthase

ROS

reactive oxygen species

SPDS

spermidine synthase

SPMS

spermine synthase

Shi HT, Ye TT, Chen FF, Cheng ZM, Wang YP, Yang PF, et al. Manipulation of arginase expression modulates abiotic stress tolerance in Arabidopsis: effect on arginine metabolism and ROS accumulation. J Exp Bot. 2013 doi: 10.1093/jxb/ers400.