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There are two specific research objectives in this proposal:
1. Having determined that a number of chlorophyll-related metabolites in soybeans (Glycine max) in both drought-susceptible, Pana (PI 597387), and drought-tolerant (PI 567731) cultivars change in abundance, and that several of these are previously unknown, establish the identities of these novel biological metabolites.
2. Having identified these chlorophyll-related metabolites in objective 1, based on the modifications to the chlorophyll molecular structure, deduce the possible biological pathways which may be impacted to produce them, thereby gleaning additional insight into molecular adaptation mechanisms to drought.

1. Molecular identities of chlorophyll-related metabolites in both drought-susceptible and drought-tolerant cultivars of soybeans; these molecular identities are currently unknown, but from tandem mass spectrometry evidence are known to contain the core structure of chlorophyll.
2. Based on the results obtained for the first deliverable, we want to identify possible biological pathways which may be impacted to produce these novel chlorophyll-related metabolites and further our understanding of molecular adaptations the soybean plant undergoes to adapt to drought stress.

Updated April 30, 2022:

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Updated July 27, 2022:

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Updated October 28, 2022:
This quarter's progress report is in the form of a draft manuscript which will be submitted to the journal Mass Spectrometry Letters in the following month.

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Updated January 30, 2023:

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Using methanolic leaf extracts from two different cultivars of soybeans, we were able to examine at the level of metabolites some of the differences between a drought-susceptible cultivar, Pana, vs. a drought-tolerant cultivar, PI 567731. Leaf extracts from the two cultivars grown under control conditions share 60 ionic formulas which are matched to the SoyCyc database. Prominent amongst these are mono- and diacylglycerols, pheophytin a and chlorophyll a, monosaccharides, disaccharides, xanthins, and vicenin-2 (a flavonoid diglucosylation product). Notable also is the simultaneous presence of plastaquinone, detected with products echinone and plastoquinol, essential components of photosynthetic electron transfer.

More interesting, however, are the metabolites which are detected in only one of the two cultivars. There are several carboxylic acid molecules present in Pana that were not detected in PI 567731; these are essential precursors to lipids. Carlactone is an oxidation product of cartenal, possibly indicating oxidative stress in Pana even in the control which has not experienced drought. This is further supported by the presence of glutathione disulfide, the oxidized dimer of glutathione. Galactopinitols are required substrates and products of galactosylcyclitol biosynthesis. The compound 15-cis-phytoene is needed for production of plastoquinol and carotenes. Likewise, the substance menoquinol-8 is a polyprenyl quinone required for electron transport. A richer more diverse complement of pheophytins and chlorophylls are detected in Pana in comparison to PI 567731 (e.g., chlorophyll b was only detected in Pana). However, our earlier work showed that PI 567731 maintains greater levels of pheophytins and chlorophylls during drought itself.

There are five metabolites uniquely detected in PI 567731 leaf extracts. First is3-ß-D-galactosyl-sn-glycerol, formed from the degradation of diacyl glycerols. Soyasapogenol B is a key precursor in the formation of its glucuronide. There are many possible structures for the trisaccharides, so anabolism of more complex saccharides from mono- and disaccharides might explain the appearance of trisaccharides here. Plastoquinones are electron carriers that are necessary building blocks for plastoquinol, and are found in chloroplasts, thus playing a central role in the photosynthetic electron transport chain. Moreover, for the metabolites unique to PI 567731, several key biosynthetic pathways are identified: saponin, glycherretinate, B series fagopyritols, starch, and stachyose. Degradation pathways include those for glycerodiphosphoesters and stachyose. One transport pathway—acquisition of phosphate—is also apparent in a metabolite specific to PI 567731. Although less chemically diverse, the metabolites identified uniquely in PI 567731 tend to be building blocks of complex sugars as well as phosphate acquisition. It may be that PI 567731 has a larger reservoir of energy storage molecules than Pana, which may enable it to adapt better to conditions of drought.

Abiotic stresses such as drought can significantly decrease soybean (Glycine max) yields, and current climate models suggest that drought will become increasingly common in all regions of the United States. The development of drought-resistant soybean cultivars depends on an improved understanding of stress-response mechanisms. While it is understood that drought stress impacts photosynthesis in soybeans, how this is manifested in the actual metabolic products observed in the plant is largely unknown. Previous support allowed us to compile catalogs of of two plant introductions (PIs) of soybeans, one which is drought-susceptible, Pana (PI 597387), and one which is drought-tolerant, (PI 567731). Catalogs of leaf metabolites for both cultivars have been generated, both for young leaves (1 week) and old (2-3 weeks) and abundances of these metabolites have been obtained. In Pana, drought had a substantial effect on a large number of metabolites within the young plant leaf extracts, almost exclusively expressed at higher level in leaves exposed to drought. Of those that showed 2x or more abundance in drought-treated leaves include hexose monosaccharides, 1-18:3,-2-18:3-monogalactosyldiacylglycerol, pheophytin a, chlorophyll a and a number of chlorophyll-related metabolites. It appears as water starts to become scarce, most metabolites, including the chlorophylls, are elevated in early stages. However, drought had a relatively small effect on most metabolites as it progresses; in the old Pana leaf extracts, only five metabolites changed in relative abundance by more than a factor of 2, metabolites with m/z 349.178, 367.188, 635.439, 787.520, and 813.491, with the first four decreasing upon drought. Only one metabolite within the Pana cultivar increased in abundance (2.6x) in the old leaves with drought-treatment: solanesyl diphosphoric acid. This metabolite is involved in biosynthesis of polyisoprenoids and nonaprenyl diphosphate, suggesting a primary mechanism by which the Pana cultivar adapts to drought conditions is to increase its production of isoprenoid lipids, possibly to minimize loss of water by producing more wax on the leaf surface. Also depletion of chlorophyll a and its related metabolites stabilizes with prolonged drought. Although Pana is a drought-susceptible cultivar, its metabolite levels become stabilized.

For the drought-tolerant cultivar, PI 567731, the metabolomics data reveals a more complex situation. Drought had a significant impact on many metabolites within the young plant leaf extracts; a 2x greater increase in abundance was observed for over 40 metabolites, including C7H14O6 (24 isomeric structures possible), 3-beta-D-galactosyl-sn-glycerol, isoorientin, kaempferol 3-O-beta-D-glucosyl-(1->2)-beta-D-glucoside, bis(beta-D-glucosyl) crocetin, 1-18:3,-2-18:3-monogalactosyldiacylglycerol, and chlorophylls a and b. Of those which indicated decreased abundance, only the peak at m/z 803.574, which corresponds to 3-methoxy-4-hydroxy-5-all-trans-nonaprenylbenzoic acid could be identified. It appears in the PI 567731 cultivar, as water starts to become scarce, most metabolites, including the chlorophylls, are elevated in early stages of drought. Unlike Pana, for the PI 567731 cultivar, drought had a substantial effect on many metabolites within the old plant leaf extracts as well; indeed, drought induced a 2x or greater decrease in the relative abundance of 55 metabolites, including bis(beta-D-glucosyl) crocetin, a plastoquinone (involved in trans-lycopene synthesis), 3-methoxy-4-hydroxy-5-all-trans-nonaprenylbenzoic acid, solanesyl diphosphoric acid, pheophytin a, chlorophyll a, and 1-18:3,-2-18:3-digalactosyldiacylglycerol. Only a single metabolite, that referred to as chlorophyll-related metabolite 1073 (m/z 1073.61), increased in abundance as a consequence of drought. Interestingly this same metabolite is reduced in abundance in the drought-induced young leaf extracts. This suggests one mechanism of adaptation of this drought-tolerant cultivar of soybeans is to shift from chlorophyll a to the chlorophyll-related metabolite 1073. Identification of this species becomes more important, as it implicates a mechanism by which a drought-tolerant cultivar is able to adapt to long periods of drought. Overall, the drought-tolerant cultivar, PI 567731, sees a greater change in metabolite levels as drought progresses. Indeed, solanesyl diphosphoric acid actually decreases with time, so PI 567731 does not adapt to drought in the same way as Pana; indeed, chlorophyll and its related metabolites appear to shift in favor of chlorophyll-related metabolite 1073 (m/z 1073.61).