T6P (trehalose 6-phosphate), the precursor of trehalose, has come out of obscurity over 10 years to be appreciated as an important regulator of plant metabolism and development, quite possibly linking the two. This information has been gained from analysis of mutant and transgenic plants, which show strong, diverse and strategically important phenotypes. Plant genes that encode the trehalose pathway are numerous and highly regulated transcriptionally and post-translationally, responding sensitively to the environment in a developmentally programmed and tissue-specific manner further suggestive of a vital function. Yet the precise role of T6P has not been clear. In an article published in the Biochemical Journal in 2006, John Lunn and colleagues addressed a major obstacle to understanding the function of T6P through development of a method capable of resolving femtomolar quantities of T6P from very small amounts of tissue. Using this technology, the authors showed large changes in T6P content that reflect tissue sucrose status. Overall, this elegant work makes an important contribution towards our understanding of the function of T6P in plants.
In contrast with the rest of plant carbohydrate metabolism, progress in understanding trehalose biology has taken quite a different course. For sucrose, starch, maltose, hexoses, fructans and raffinoses, the major non-structural carbohydrates of the plant kingdom, progress was facilitated because carbohydrate profiles through enzymatic and chromatographic separation techniques were readily determined. It was possible to analyse how these pools were affected by the environment during plant development in different tissues and species. Reaction sequences were determined, enzymes responsible for their metabolism were purified and characterized, the genes were cloned and transgenic plants were made. Subsequent availability of the complete genome sequence of Arabidopsis thaliana confirmed and refined information that was already available.
Trehalose, in contrast, evaded enquiry until recently because it did not show up in conventional analytical procedures, the exception being a few obscure curiosity species and in plant–fungal interactions in root nodules. For trehalose, the beginning of an appreciation of its more central importance in plants was initiated through the creation of transgenic plants expressing Escherichia coli genes for the pathway which were generated with the idea to utilize plants as a vehicle for trehalose production and to examine any possible side effects on stress tolerance . These transgenic plants showed quite dramatic and different phenotypes from those produced through modification of enzymes involved in the metabolism of other carbohydrates. They included beneficial effects such as increased photosynthetic capacity, a trait that had previously been difficult to improve . Subsequent availability of the full A. thaliana genome revealed a total of 21 genes putatively involved in the synthesis of trehalose: 11 TPSs (trehalose phosphate synthases) and ten TPPs (trehalose phosphate phosphatases) . This is more than the number of genes found for the synthesis of other plant carbohydrates.
Some conclusions could be drawn about the function of the trehalose pathway from these studies, even in the absence of high-resolution detection methods. First, the function of trehalose in plants was certainly likely to be different from that of other sugars. Sucrose, the other widespread non-reducing disaccharide, is found in large amounts in plants. This enables it to function as translocated carbon source, particularly as a ready source of carbon for cell wall synthesis and starting point for all other organic compounds in growing tissues. Its non-reducing properties and accumulation to high levels also suit a function as stress protectant. Trehalose, whose properties are similar, could not realistically perform analogous functions in such low amounts. However, in combination with proteins known as late-embryogenesis-abundant proteins, there is the likelihood that trehalose has a function in drought stress tolerance [4,5]. Molecules in low abundance typically function as signals (gibberellic acid, for example). It is striking that, unlike those involved in the sucrose pathway, the genes involved in the synthesis of trehalose cluster around the intermediate T6P (trehalose 6-phosphate), which could indicate that the regulation of the content of T6P is particularly important and not just for the synthesis of trehalose. Overall, a hypothesis could be presented that T6P was a sugar signal whose synthesis and degradation was carefully regulated for co-ordinated regulation of pathways in response to prevailing carbon supply, of, for example, photosynthesis .
Before the new method of Lunn et al.  appeared in the Biochemical Journal, methods that had been used to measure sugars that were found in millimolar amounts in biological material were adapted to quantify T6P and trehalose. These included an HPLC method [8,9] and inhibition of Yarrowia lipolytica hexokinase [2,10]. None was ideal, given the low abundance of the compounds that they were trying to detect. Much painstaking sample preparation and replication was necessary to achieve reproducible estimates. The new method of Lunn represented an important advance. A simple extraction procedure for T6P was devised using chloroform, methanol and finally water, with a filtering procedure to remove viscous high-molecular-mass components yielding good recoveries of T6P. Subsequent analysis then uses anion-exchange HPLC in tandem with LC (liquid chromatography), which separates T6P from other compounds, and then identifies and quantifies it with LC–MS-Q3 (triple-quadrupole MS), combining resolution with exceptional detection sensitivity. The authors further used TPP and sucrose phosphate phosphatase to specifically remove T6P and sucrose 6-phosphate respectively, compounds of the same mass, to further specify peak assignment. Overall, the resolving powers of this technology are 100-fold greater than those of previous methods. In conclusion, the analysis was able to show a large dynamic range (27-fold) of T6P of 18–482 pmol·g−1 of fresh weight in seedlings and leaves of A. thaliana grown under low-light (130 μmol of quanta·m−2·s−1) 12-h day growing conditions and in response to treatments of extended night and 15 mM sucrose feeding, as well as in a starchless mutant. The authors calculated tissue concentrations in the range 1–15 μM.
These estimates are likely to be the most accurate direct determinations of T6P published to date. They are in the region of 5-fold lower than other published values. There are a number of possible reasons for this difference. First, it is possible that earlier methods were not able to adequately distinguish T6P from other compounds. Secondly, T6P has a large dynamic range that responds strongly to carbon supply. Growing conditions used by Lunn et al.  were at the lower end of carbon availability, i.e. low light, 12-h day, that plants experience under natural conditions. In contrast, in the other published work, higher light, longer days and higher sucrose (or 100 mM trehalose ) were used. Given that TPS and TPP genes respond rapidly and strongly to the environment, and particularly to carbon status, T6P levels could have been higher in earlier studies in reflection of this. Thirdly, it is likely that there are differences between species and that too many conclusions on absolute levels of T6P should not be drawn from a restricted number of measurements on one species. Significantly, in recent work on rice , TPP1 and TPP2 Km values of 92 and 186 μM T6P respectively were shown. Km values for enzymes fall within the range of actual in vivo metabolite concentrations, consistent with higher concentrations of T6P and quite possibly an even wider dynamic range of T6P than reported by Lunn et al. .
An overall important conclusion of the work is the strong correlation between T6P and sucrose content. This does present and confirm the possibility that sugar-induced changes in T6P play a central role in carbohydrate utilization  and storage  in plants in the context of prevailing carbon availability. Why should sucrose need a specific signal to communicate its availability when sucrose can and does convey information directly ? T6P would provide the ability to supply a further level of communication that could integrate information on sucrose supply with other factors such as environmental stress (trehalose pathway genes are strongly regulated by stress as well as by sugar ) to elicit the appropriate response under the prevailing sucrose status for those particular conditions. For example, high sucrose under low soil nitrogen or phosphorus could lead to different signalling outcomes. Furthermore, as T6P is not part of a major metabolite flux to an important end-product, T6P levels can fluctuate without compromising other functions, as would happen for central metabolites such as G6P (glucose 6-phosphate) and UDPG (UDP-glucose), from which T6P is synthesized. Potentially T6P could signal availability of these central intermediates. The small pool size of T6P means that turnover times are high, which would facilitate rapid responses to vital functions that require rapid adjustment.
The Lunn work is an important contribution to the still-emerging field of trehalose biology in plants. Further important steps forward will include knowledge of the localization of T6P in specific cell types and of downstream targets to disclose the vital function of T6P. The role of the large number of trehalose pathway genes will need to be elucidated and how they control T6P synthesis and breakdown. Given the range of important phenotypes associated with T6P, it is likely that modification of T6P signalling has been selected for in crop improvement, e.g. the ramosa3 mutant of maize . Further selection and targeting of T6P signalling to specific cells is likely to bring additional benefit, guided by knowledge of downstream targets of T6P.
Rothamsted Research receives grant-aided support from the Biotechnological and Biological Sciences Research Council (BBSRC) of the U.K. M. P. acknowledges BBSRC grants BB/C51257X/1 and BB/D006112/1.