Cysteine proteases and wheat (Triticum aestivum L) under drought: A still greatly unexplored association

Bread wheat (Triticum aestivum L.) provides about 19% of global dietary energy. Environmental stress, such as drought, affects wheat growth causing premature plant senescence and ultimately plant death. A plant response to drought is an increase in protease‐mediated proteolysis with rapid degradation of proteins required for metabolic processes. Among the plant proteases that are increased in their activity following stress, cysteine proteases are the best characterized. Very little is known about particular wheat cysteine protease sequences, their expression and also localization. The current knowledge on wheat cysteine proteases belonging to the five clans (CA, CD, CE, CF and CP) is outlined, in particular their expression and possible function under drought. The first successes in establishing an annotated wheat genome database are further highlighted which has allowed more detailed mining of cysteine proteases. We also share our thoughts on future research directions considering the growing availability of genomic resources of this very important food crop. Finally, we also outline future application of developed knowledge in transgenic wheat plants for environmental stress protection and also as senescence markers to monitor wheat growth under environmental stress conditions.     

Plant cysteine proteinases: evaluation of the pharmacological activity

Cysteine proteinases are involved in virtually every aspect of plant physiology and development. They play a role in development, senescence, programmed cell death, storage and mobilization of germinal proteins, and in response to various types of environmental stress. In this review, we focus on a group of plant defensive enzymes occurring in germinal tissue of Caricaceae. These enzymes elicit a protective response in the unripe fruit after physical stress. We propose that these enzymes follow a strategy similar to mammalian serine proteinases involved in blood clotting and wound healing. We show evidence for the pharmacological role of plant cysteine proteinases in mammalian wound healing, immunomodulation, digestive conditions, and neoplastic alterations.     

Vacuolar cysteine proteases of wheat (Triticum aestivum L.) are common to leaf senescence induced by different factors

Cellular proteins are extensively degraded during leaf senescence, and this correlates with an up-regulation of protease gene expression, particularly cysteine proteases. The objectives of this work were (i) to detect cysteine proteases associated with senescence of wheat leaves under different conditions and (ii) to find out their subcellular location. Activity labelling of cysteine proteases with the biotinylated inhibitor DCG-04 detected five bands at 27, 36, 39, 42, and 46 kDa in leaves of wheat senescing under continuous darkness. In-gel activity assays showed that these proteases are only active in an acid milieu (pH 4), and their activity increased several-fold in senescing leaves. Fractionation experiments showed that the senescence-associated cysteine proteases of 36, 39, 42, and 46 kDa localize to a vacuolar-enriched fraction. The vacuolar cysteine proteases of 36, 39, and 42 kDa increased in activity in attached flag leaves senescing naturally during post-anthesis, and in attached leaves of plants subjected to a period of water deficit. Thus, the activity of these vacuolar cysteine proteases is associated with developmental (post-anthesis) senescence and with senescence induced by stress factors (i.e. protracted darkness or drought). This suggests that vacuoles are involved in senescence-associated cellular degradation, and that different senescence-inducing factors may converge on a single degradation pathway.     

A nodule-specific plant cysteine proteinase, AsNODF32, is involved in nodule senescence and nitrogen fixation activity of the green manure legume Astragalus sinicus

Asnodf32, encoding a nodule-specific cysteine proteinase in Astragalus sinicus, is probably involved in nodule senescence. To obtain direct evidence of its role in nodule senescence, Agrobacterium rhizogenes-mediated RNA interference was applied to A. sinicus hairy roots. Real-time qRT-PCR was used to estimate the efficiency of suppression. The senescent phenotype of transgenic nodules was examined with paraffin-embedded slides, TUNEL (TdT-mediated dUTP nick-end labeling) assay, and transmission electron microscopy, and the bacteroid nitrogen fixation activity was also measured. It was found that silencing of Asnodf32 delayed root nodule and bacteroid senescence. The period of bacteroid active nitrogen fixation was significantly extended. Interestingly, nodules enlarged in length were also observed on Asnodf32-silenced hairy roots. The results reported here indicate that Asnodf32 plays an important role in the regulation of root nodule senescence.     

The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants

Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhibitors was ineffective. A glutathione S-transferase-cystatin fusion protein was used to purify and characterize the induced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants.     

Ectopic phytocystatin expression leads to enhanced drought stress tolerance in soybean (Glycine max) and Arabidopsis thaliana through effects on strigolactone pathways and can also result in improved seed traits

Ectopic cystatin expression has long been used in plant pest management, but the cysteine protease, targets of these inhibitors, might also have important functions in the control of plant lifespan and stress tolerance that remain poorly characterized. We therefore characterized the effects of expression of the rice cystatin, oryzacystatin‐I (OCI), on the growth, development and stress tolerance of crop (soybean) and model (Arabidopsis thaliana) plants. Ectopic OCI expression in soybean enhanced shoot branching and leaf chlorophyll accumulation at later stages of vegetative development and enhanced seed protein contents and decreased the abundance of mRNAs encoding strigolactone synthesis enzymes. The OCI‐expressing A. thaliana showed a slow‐growth phenotype, with increased leaf numbers and enhanced shoot branching at flowering. The OCI‐dependent inhibition of cysteine proteases enhanced drought tolerance in soybean and A. thaliana, photosynthetic CO₂ assimilation being much less sensitive to drought‐induced inhibition in the OCI‐expressing soybean lines. Ectopic OCI expression or treatment with the cysteine protease inhibitor E64 increased lateral root densities in A. thaliana. E64 treatment also increased lateral root densities in the max2‐1 mutants that are defective in strigolactone signalling, but not in the max3‐9 mutants that are defective in strigolactone synthesis. Taken together, these data provide evidence that OCI‐inhibited cysteine proteases participate in the control of growth and stress tolerance through effects on strigolactones. We conclude that cysteine proteases are important targets for manipulation of plant growth, development and stress tolerance, and also seed quality traits.     

The roles of redox processes in pea nodule development and senescence

Nodule senescence is triggered by developmental and environmental cues. It is orchestrated through complex but poorly characterized genetic controls that involve changes in the endogenous levels of reactive oxygen species (ROS) and antioxidants. To elucidate the importance of such redox control mechanisms in pea root nodule senescence, redox metabolites were analysed throughout nodule development in a commercial pea variety ( Pisum sativum cv. Phoenix) inoculated with a commercial rhizobial strain ( Rhizobium leguminosarum bv. viciae ). Although a strong positive correlation between nitrogenase activity and nodule ascorbate and glutathione contents was observed, the progressive loss of these metabolites during nodule senescence was not accompanied by an increase in nodule superoxide or hydrogen peroxide. These oxidants were only detected in nodule meristem and cortex tissues, and the abundance of superoxide or hydrogen peroxide strongly declined with age. No evidence could be found of programmed cell death in nodule senescence and the protein carbonyl groups were more or less constant throughout nodule development. Pea nodules appear to have little capacity to synthesize ascorbate de novo . l -galactono-1, 4-lactone dehydrogenase (GalLDH), which catalyses the last step of ascorbate synthesis could not be detected in nodules. Moreover, when infiltrated with the substrates l -galactono-1, 4-lactone or l -gulonolactone, ascorbate did not accumulate. These data suggest that ROS, ascorbate and glutathione, which fulfil well recognized, signalling functions in plants, decline in a regulated manner during nodule development. This does not necessarily cause oxidative stress but rather indicates a development-related shift in redox-linked metabolite cross-talk that underpins the development and aging processes.     

Developmental fate of Rhizobium meliloti bacteroids in alfalfa nodules.

Nitrogen-fixing bacteroids are degraded during nodule senescence. This is in contrast to recent implications that viable bacteroids can be released into soil from legume nodules. Rhizobia originating from persistent infection threads in senescing nodule plant cells seem to be the source of viable cells required for perpetuation of the Rhizobium spp. population in the soil. Our conclusions were derived from electron microscopic examination of stages of development and senescence of alfalfa root nodules.     

Isolation and characterization of a root nodule-specific cysteine proteinase cDNA from soybean

We have determined that a nodule-specific cDNA clone (GmCysP1), obtained from a soybean root nodule-specific EST pool, encodes cysteine proteinase. Its amino acid sequence homology, as well as the conservation of typical motifs and amino acid residues involved in active site formation, shows that GmCysP1 can be classified as a legumain (C13) family cysteine proteinase, belonging to clan CD. Moreover, based on its expression patterns,GmCysP1 is a nodule-specific cysteine proteinase gene that is possibly associated with nodule development or senescence. Our genomic Southern analysis also suggests thatGmCysP1 is a member of a multigene family. Therefore, we propose that GmCysP1 is the first to be identified as a nodule-specific and senescence-related cysteine proteinase that belongs to the legumain family from soybean.     

Engineering and genetic approaches to modulating the glutathione network in plants

Reduced glutathione (GSH) is the most abundant low-molecular weight thiol in plant cells. It accumulates to high concentrations, particularly in stress situations. Because the pathway of GSH synthesis consists of only two enzymes, manipulation of cellular glutathione contents by genetic intervention has proved to be relatively straightforward. The discovery of a new bacterial bifunctional enzyme catalysing GSH synthesis but lacking feedback inhibition characteristics offers new prospects of enhancing GSH production and accumulation by plant cells, while the identification of γ-glutamyl cysteine and glutathione transporters provides additional possibilities for selective compartment-specific targeting. Such manipulations might also be used to affect plant biology in disparate ways, because GSH and glutathione disulphide (GSSG) have crucial roles in processes as diverse as the regulation of the cell cycle, systemic acquired resistance and xenobiotic detoxification. For example, depletion of the total glutathione pool can be used to manipulate the shoot : root ratio, because GSH is required specifically for the growth of the root meristem. Similarly, chloroplast γ-glutamyl cysteine synthetase overexpression could be used to increase the abundance of specific amino acids such as leucine, lysine and tyrosine that are synthesized in the chloroplasts. Here we review the aspects of glutathione biology related to synthesis, compartmentation and transport and related signalling functions that modulate plant growth and development and underpin any assessment of manipulation of GSH homeostasis from the viewpoint of nutritional genomics.     

Plant Cell Wall Remodelling in the Rhizobium–Legume Symbiosis

Colonization of host cells by rhizobium bacteria involves the progressive remodelling of the plant–microbial interface. Following induction of nodulation genes by legume-derived flavonoid signals, rhizobium secretes Nod-factors (lipochitin oligosaccharides) that cause root hair deformations by perturbing the growth of the plant cell wall. The infection thread arises as a tubular ingrowth bounded by plant cell wall. This serves as a conduit for colonizing bacterial cells that grow and divide in its lumen. The transcellular orientation of thread growth is controlled by the cytoskeleton and is coupled to cell cycle reactivation and cell division processes. In response to rhizobium infection, host cells synthesize several new components (early nodulins) that modify the properties of the cell wall and extracellular matrix. Root nodule extensins are a legume-specific family of hydroxyproline-rich glycoproteins targeted into the lumen of the infection thread. They have alternating extensin and arabinogalactan (AGP) glycosylation motifs. The structural characteristics of these glycoproteins suggest that they may serve to regulate fluid-to-solid transitions in the extracellular matrix. Extensibility of the infection thread is apparently controlled by peroxide-driven protein cross-linking and perhaps also by modification of the pectic matrix. Endocytosis of rhizobia from unwalled infection droplets into the host cell cytoplasm depends on physical contact between glycocalyx components of the plant and bacterial membrane surfaces. As endosymbionts, bacteroids remain enclosed within a plant-derived membrane that is topologically equivalent to the plasma membrane. This membrane acquires specialist functions that regulate metabolite exchanges between bacterial cells and the host cytoplasm. Ultimately, however, the fate of the symbiosome is to become a lysosome, causing the eventual senescence of the symbiotic interaction.     

A nodule-specific gene encoding a subtilisin-like protease is expressed in early stages of actinorhizal nodule development.

To identify genes specifically expressed during early stages of actinorhizal nodule development, a cDNA library made from poly(A) RNA from root nodules of Alnus glutinosa was screened differentially with nodule and root cDNA, respectively. Seven nodule-enhanced and four nodule-specific cDNA clones were isolated. By using in situ hybridization, two of the nodule-specific cDNAs were shown to be expressed at the highest levels in infected cells before the onset of nitrogen fixation; one of them, ag12 (A. glutinosa), was examined in detail. Sequencing showed that ag12 codes for a serine protease of the subtilisin (EC 3.4.21.14) family. Subtilisins previously appeared to be limited to microorganisms. However, subtilisin-like serine proteases have recently been found in archaebacteria, fungi, and yeasts as well as in mammals; a plant subtilisin has also been sequenced. In yeast and mammals, subtilases are responsible for processing peptide hormones. A homolog of ag12, ara12, was identified in Arabidopsis; it was expressed in all organs, and its expression levels were highest during silique development. Hence, our study shows that subtilases are also involved in both symbiotic and nonsymbiotic processes in plant development.     

Redox regulation of peroxiredoxin and proteinases by ascorbate and thiols during pea root nodule senescence

Redox factors contributing to nodule senescence were studied in pea. The abundance of the nodule cytosolic peroxiredoxin but not the mitochondrial peroxiredoxin protein was modulated by ascorbate. In contrast to redox-active antioxidants such as ascorbate and cytosolic peroxiredoxin that decreased during nodule development, maximal extractable nodule proteinase activity increased progressively as the nodules aged. Cathepsin-like activities were constant throughout development but serine and cysteine proteinase activities increased during senescence. Senescence-induced cysteine proteinase activity was inhibited by cysteine, dithiotreitol, or E-64. Senescence-dependent decreases in redox-active factors, particularly ascorbate and peroxiredoxin favour decreased redox-mediated inactivation of cysteine proteinases.