NO way to die – nitric oxide, programmed cell death and xylogenesis
Centre for Research in Plant Science, Faculty of Applied Sciences, University of the West of England, Bristol BS16 1QY, UK (tel +44 (0) 117 3282149; fax +44 (0) 117 3282132; email Steven.Neill@uwe.ac.uk)
Xylem cells have to be killed – the mature elements are terminally differentiated cells that have undergone secondary cell wall thickening and lignification coupled with programmed cell death (PCD) that results in the formation of rigid hollow tubes specialised for water transport. PCD occurs in various forms throughout the plant life-cycle, probably with both common and specific aspects (Lam, 2004). Nitric oxide (NO) is a small reactive gas for which signalling roles in an increasing number of biological processes, including PCD, are emerging. Plant cells generate NO in response to challenge by microbial pathogens or elicitors derived from them and, probably in concert with other signals such as reactive oxygen species, mediate the pathogen-induced PCD that occurs during the hypersensitive response (HR) (Neill et al. 2003; Wendehenne et al. 2004). Exciting data published in this issue by Gabaldon et al. (pp. 121–130) bring together these topics and demonstrate that NO is also a key factor regulating PCD and lignification during xylem formation.
‘These data indicate strongly that NO is a key factor mediating PCD and lignification during xylogenesis’
The Zinnia elegans system
Gabaldon et al. use Zinnia elegans as their model species and use two complementary experimental systems – developing vascular bundles in stems of Zinnia seedlings and a remarkable in vitro Zinnia cell culture system often used to study xylogenesis (Roberts & McCann, 2000). In the first system, confocal laser scanning microscopy of stem sections incubated with an NO-sensitive fluorescent dye revealed that NO production was largely confined to xylem cells (and, incidentally, phloem cells that also undergo lignification). Moreover, Gabaldi et al. observed a spatial gradient of NO production inversely related to the degree of lignification, with the highest fluorescence being seen in just-differentiating cells and the lowest in differentiated, lignified cells.
These observations in planta were mirrored by those made using the in vitro cell culture. In this second system, mesophyll cells are isolated from leaves and incubated in a hormone-containing medium that induces the transdifferentiation of the cells into tracheary elements. Here, Gabaldon et al. found that there was a temporal gradient of NO production, with low NO-fluorescence in the undifferentiated cells, the highest NO-fluorescence in differentiating thin-walled cells and then low NO-fluorescence again in thick-walled differentiated cells. That NO is essential for both differentiation and PCD was demonstrated by the effects of the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline (PTIO), which very effectively removes NO from cells both in planta and in culture. Removal of NO from the cultured cells with PTIO resulted in dramatic reductions in both PCD and the formation of tracheary elements.
Although absolute verification will require genetic and transgenic analyses, together these data indicate strongly that NO is a key factor mediating PCD and lignification during xylogenesis. In addition, NO might well directly affect the activity of some of the enzymes of lignin biosynthesis as well as increasing the transcription of their genes (see Gabaldon et al.). Transcriptional profiling of transdifferentiating Zinnia cells has identified many genes induced or repressed during xylogenesis (Demura et al., 2002); profiling in the presence of PTIO could be an excellent way to determine which genes are regulated directly by NO.
Where is the nitric oxide from?
Although the data reported by Gabaldon et al. do indicate the importance of NO, it remains to be seen how NO is made in developing xylem cells and how its concentrations are regulated. Significant advances have been made recently with regard to the sources of NO in plants, with nitrate reductase (NR) and two different nitric oxide synthase (NOS) enzymes (iNOS and AtNOS1) being characterised (Fig. 1; see Wendehenne et al., 2004). However, inhibitor studies carried out by Gabaldi et al. suggest that none of these enzymes is responsible for xylem-produced NO – clearly another exciting avenue to be explored.
Other factors in the pathway
Cellular responses to NO include transient elevations in cytosolic calcium (Fig. 1; Wendehenne et al., 2004), a process also required for xylem PCD (see Lam, 2004). It seems likely that in plants, as in animals, NO activates the enzyme guanyl cyclase, generating the second messenger cGMP (Fig. 1; see Neill et al., 2003; Pagnussat et al., 2003). cGMP could activate calcium channels directly, or indirectly, via formation of the calcium channel regulator cyclic ADP-ribose or through stimulation of protein phosphorylation cascades (Fig. 1). Additional aspects of NO signalling might involve activation of mitogen-activated protein kinases (MAPKs; see Neill et al., 2003; Pagnussat et al., 2004) and changes in the conformation and thus activity of NO target proteins via S-nitrosylation or tyrosine nitrosation (Fig. 1).
The precise mechanisms by which xylem PCD is executed are not yet known. Although PCD in animal cells is associated with the activation and action of caspases, cysteine proteases with discrete targets, no true caspases have been identified in plants. However, plants do possess caspase-like protease (CLP) activity and contain a range of proteases that potentially fulfil caspase functions, such as vacuolar processing enzymes (VPEs), serine proteases and metacaspases (Lam, 2004; Woltering, 2004). In fact, xylem vacuoles and cells contain many proteases and xylem PCD does indeed require the action of cysteine and serine protease (Roberts & McCann, 2000; Lam, 2004). Recent reports have shown that VPEs do possess caspase activity (Hatsugai et al., 2004; Rojo et al., 2004), that UV-induced PCD in Arabidopsis requires the action of a CLP (Danon et al., 2004) and that PCD of suspensor cells in developing Picea abies somatic embryos is dependent on the expression and action of a metacaspase (Suarez et al., 2004). Intriguingly, the expression of this Picea caspase, mcIIPa, was also seen in pro-cambial strands, the early stage of xylem development.
In summary, the data reported in this issue by Gabaldon et al. demonstrate an essential signalling role for NO during xylem PCD. Other forms of PCD similarly involve NO, such as the bacterium- or elicitor-induced HR in various systems, in some of which a requirement for CLP activity is known (Clarke et al., 2000; Wendehenne et al., 2004), in addition to hypoxia-induced root cell death (Dordas et al., 2003). These data should pave the way for future studies using both the Zinnia and Arabidopsis genetic systems to elucidate aspects of NO and PCD signalling during xylem formation.