This study shows that under natural conditions Arabidopsis plants respond to changes in their FDE capacity quite dramatically. Our initial assumption was that a lack of PsbS, and hence FDE, would reduce the plants' Darwinian fitness as a consequence of changes in their photosynthetic performance. This may be true in part , but this study demonstrates that the plant's "core metabolism" (carbohydrate and amino acid metabolism) is also strongly affected, with large changes to the transcriptome occurring in plants lacking PsbS. Several amino acids (e.g. glutamine, glycine, aspartate and threonine) were highly depleted in plants lacking PsbS, whereas levels of others (e.g. leucine) were higher. Various carbohydrates also accumulated in plants lacking PsbS including (inter alia) galactose, raffinose and isomaltose, while sucrose levels were reduced. Knowledge of metabolic changes induced in Arabidopsis by different treatments is rather limited, but several of these compounds have been reported to accumulate under various kinds of stresses. For instance, there have been suggestions that the raffinose family oligosaccharides (RFOs) provide tolerance against drought, high salinity and cold stress . Hence, the metabolic reprogramming that occurred in plants lacking PsbS could be described in general terms as a stress response. Since transcriptional changes induced in Arabidopsis by various treatments have been intensively examined in several large-scale DNA microarray projects, they are far better characterized than overall metabolome changes. This allowed us to compare our transcriptome data with responses induced by a variety of biotic and abiotic stresses, as well as hormone treatments.
Comparing microarray data obtained using different platforms is far from straightforward but there are examples showing that cross-platform comparisons can give reliable results. For example, a benchmarking of GST-based CATMA arrays (which we used), and Affymetrics arrays indicated that the results were largely consistent . However, no study, including our own, has provided data on the degree of overlap between sets of transcripts in identical RNA samples identified as being upregulated in both CATMA and Affymetrics analyses. Hence, it is not easy to evaluate the absolute level of overlap between the set of genes induced in npq4 plants and those induced by other treatments, especially since our data were obtained under field conditions, while those presented in all other studies published to data were obtained from plants grown under controlled conditions in the lab. Nevertheless, the response seen in the npq4 genotype was most similar to those reportedly induced by ozone exposure or wounding, and also similar to a JA/MeJ response. The latter finding was further corroborated in four ways: comparison with other array data; detailed analysis of genes and transcripts associated with the JA/MeJa pathway in our plants; proteomic analysis; and measuring the JA levels within the plants. We hypothesize that a lack of PsbS in Arabidopsis plants leads to increased photooxidative stress and consequently induces a stress response. The signals involved are likely to be complex and to involve several hormones/signalling substances. However, since the response we found most closely resembled a JA/MeJA response in terms of the upregulated genes in microarray experiments, JA is probably one of the substances involved, and perhaps the most important. Additional studies using mutants with disturbances in different signalling pathways will hopefully provide further information regarding the nature of the signalling pathways involved.
It should be pointed out that plants probably have several mechanisms for sensing high light, and the signalling pathways involved in the responses observed in our study are unlikely to be identical to those that mediate general high light responses. For example, the effect of cryptochrome on high light-induced changes in gene expression  is apparently mediated by a different pathway from the response reported here, and the connection between ABA and high-light responses reported by  may involve different pathways too. However, it should be noted that there was some overlap between the genes induced in npq4 and those reportedly induced by ABA, although to a lower degree than the overlap seen with the JA response. Plants exposed to excessive light conditions need to modify many aspects of their growth and metabolism, since high light may be accompanied with any one or more of various other stressors, so multiple pathways could allow plants to respond to ecological challenges flexibly, with strong selective advantages.
In contrast to the abovementioned studies, here we compared genotypes that differed only in their capacity to dissipate excess light in the photosynthetic light-harvesting antenna, grown under identical environmental conditions. Thus, we focused on a single aspect of high-light signalling, mediated by signals directly generated at the photosynthetic reaction site. The results show that increased photooxidative stress at photosystem II is somehow sensed and a nuclear signal is generated that modifies gene expression. This in turn leads to a reprogramming of plant metabolism, with the implication that JA could be involved in the process. JA might even be the direct signal, and our finding that two enzymes involved in the JA biosynthesis pathway were enriched in the thylakoid membranes of PsbS-deficient plants may provide clues regarding the mechanism behind the response. Activation of the JA pathway is believed to occur at the site of JA biosynthesis; the chloroplastic membrane . Therefore, JA is locally synthesized in response to light stress, and is also amplified by a positive feedback loop . However, the level of JA was unaffected in npq4 plants unless they were provoked by herbivory. This does not exclude the direct involvement of the octadecanoid pathway in the response; increases could be induced in the flux through the pathway or in levels of either MeJA or JA-Ile, which may be the active species . However, it is also possible that plants lacking PsbS could be more "primed" and respond more actively to herbivory by increasing JA levels. At this point it is not possible to distinguish between these possibilities, but we hope to address these possibilities in future studies.
Regardless of the exact role that JA plays in the process, the metabolic reprogramming may be sufficient to explain the lower fitness observed in plants lacking PsbS, since JA-induced responses are known to reduce seed production . Furthermore, Arabidopsis plants treated with JA have an appearance that, in our experience, closely resembles that of plants exposed to moderate light stress, with anthocyanin accumulation, changes in leaf morphology and growth retardation (unpublished results). Overall, these findings suggest that plants lacking PsbS allocate resources away from reproduction towards defence. It has become increasingly apparent that stress responses are very complex. For instance, different herbivores may elicit specific responses in attacked plants and jasmonate signalling may affect different herbivores in different ways . Furthermore, although responses to various abiotic stresses share several common features, they also exhibit many highly specific features. Since most stress signalling has been shown to involve ROS in some way, one speculative possibility is that the changes we have observed could correspond to a more basal metabolic shift from reproduction and/or growth towards defence that increases the plant's ability to respond competently to stimuli elicited by specific stresses.
PsbS levels in wild-type plants do not appear to be "saturated", suggesting that selection acts against the production of higher levels of PsbS, even though it would increase the FDE capacity and hence potentially provide more photoprotection. Although this must theoretically hold true for the expression levels of all genes, it is not easy to demonstrate experimentally. An interesting implication of our data is that too much photoprotection could compromise "stress signalling", either in a more general sense, as discussed above, or more specifically through the involvement of JA/MeJA. Hence, plants with elevated PsbS levels could have less competence to cope with other stresses, and recent data indicate that this is indeed the case (Frenkel et al, in preparation). Overall, these findings suggest that there might be a tradeoff between photoprotection and tolerance to other stresses, as indicated by our data showing differences in herbivore preferences between wild-type plants and plants lacking PsbS.
Many mutants show marked phenotypic deviations from corresponding wild types, but mutations in most genes lead to more subtle changes in knock-out or antisense plants, with the lack of observable phenotypic deviations being the rule rather than the exception (see e.g. [28, 29]). Functional redundancy, of course, could be one reason for this, but it is clear that in many cases no observable phenotypic change may occur because the plants are not grown under suitable conditions for such changes to appear. Plant metabolic networks have highly complex architectures and the ability to compensate for enzyme deficiencies, or even an insufficiency in an entire pathway, using alternative pathways or substances that can perform similar functions, is likely to be of great adaptive value. Growing plants under naturally stressful conditions, rather than under controlled conditions in the lab, inevitably generates data that are complex and difficult to interpret. To our knowledge, the present study provides the first demonstration that metabolomic and transcriptomic analyses of mutants and over-expressers grown in the field can provide new and unexpected conclusions about individual gene products. We have been able to separate the genotypic differences from uncontrolled variation, and metabolomic analyses performed in the field and the laboratory clearly demonstrated that genotypic differences in expression patterns observed in field-grown material were not necessarily present under controlled conditions. It should be stressed that the responses described here were not detected under standard growth conditions in the lab. Therefore, we believe that studies like this, using plants grown under natural conditions, are essential for appreciating the full complexity of plants' genetic and metabolic composition and their interactions with the environment.