This data set explores the effect of the cyanobacterial gene ictB on photosynthesis in sorghum, under both normal greenhouse growing temperatures (32 C / 25 C) and during and after an 8 day chilling stress (10 C / 5 C). IctB is a cyanobacterial gene of unknown function, which was initially thought to be involved in inorganic carbon transport into cells. While ictB is known now not to be an independently active carbon transporter in its own right, it may play a role in passive diffusion of metabolites. This transgene was introduced into sorghum by the lab of Thomas Clemente, through Agrobacterium mediated transformation, alone and in combination with the tomato sedoheptulose-1,7-bisphosphatase (SBPase) gene. Eleven events (six double construct and five single construct ictB) were involved in this study. SBPase was included because some previous experiments in C3 species and some previous modeling work, as well as its position at a metabolic branch point, indicates it plays a role as a control point for photosynthesis. A chilling treatment was included because chilling is one of the most serious ecological factors limiting the range of C4 species.

Data includes gene expression, metabolomics (at normal growing temperature), SBPase enzyme activity, biomass and photosynthetic traits at both warm temperature and during and after chilling stress.
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EXPLANATORY NOTES FOR ICTB/SBPASE SORGHUM MANUSCRIPT

Data are organized into 10 worksheets, representing an expected 10 tables that will serve a supplementary role in the final publication. These include data on gene expression, metabolomics (at normal growing temperature), SBPase enzyme activity, biomass and photosynthetic traits at both warm temperature and during and after chilling stress.

Tables are as follows:
1. Event_Code: for Table S1. Event codes for events and constructs. Two constructs were generated for this study, and numerous transgenic “events” (i.e. independent transformations) were carried out for each construct. A construct represents the actual vector which was introduced into the plants (complete with promoter, gene of interest, marker gene, etc.) while an event represents a single successful introduction of the transgene. Events are uniquely labeled with letter and number strings but also with a four-digit number for ease of reference, this table explains which event corresponds to each four-digit number.
2. Photosynthetic_Data: for Table S2. Photosynthetic data at greenhouse growing temperature, for ictB single construct, ictB/SBPase double construct, and wild type lines. Five ictB and six ictB/SBPase events were included. Greenhouse growing temperature was approximately 32 °C and 25 °C night. Photosynthetic parameters were measured using a Licor 6400-XT, and included parameters related to carbon dioxide uptake, water loss, and chlorophyll fluorescence.
3. Chilling_Treatment: for Table S3. Photosynthetic response to chilling treatment, for ictB single construct, and wild type lines. Four ictB events were included. Chilling treatment lasted approximately 8 days and began either 3.5 or 5.5 weeks after transplanting the plants (chilling was done in two batches). Chilling treatment involved temperatures of 10 °C day / 7 °C night in growth chambers. Photosynthetic parameters were measured at several time points during and after the chilling treatment, were measured using a Licor 6400-XT, and included parameters related to carbon dioxide uptake, water loss, and chlorophyll fluorescence.
4. SBPase_Activity: for Table S4. SBPase activity in double construct plants. These data measure in vitro substrate-saturated activity of SBPase in desalted extracts from leaf tissues, at 25 °C. Units are micromoles of SBP processed per second per m2 of leaf tissue. Five ictB/SBPase events were included.
5. 2014_gene_exp: for Table S5. Gene expression in 2014 experiment (units of cycle times). These data measure cycle times to threshold, relative to reference genes, for expression of ictB and SBPase. Six ictB single construct events and five ictB/SBPase double construct events were included. Cycle times to threshold relative to reference genes (ΔCT) are inversely related to number of transcripts relative to reference genes, as follows:

ΔCT = -log2([NictB]/[Nreference])/[1 + log2b] where b = efficiency of replication.

6. 2016_gene_exp: for Table S5. Gene expression in 2016 experiment (units of cycle times). These data measure cycle times to threshold, relative to reference genes, for expression of ictB and SBPase. Six ictB single construct events and five ictB/SBPase double construct events were included. Cycle times to threshold relative to reference genes (ΔCT) are inversely related to number of transcripts relative to reference genes, as follows:

ΔCT = -log2([NictB]/[Nreference])/[1 + log2b] where b = efficiency of replication.

7. Metabolites: for Table S7. Levels of 267 metabolites in leaf tissue. Four ictB single construct events and four ictB/SBPase double construct events were included in these analyses. Metabolites were measured in methanol-extracted samples, either by liquid chromatography / mass spectrometry or by gas chromatography / mass spectrometry, and were compared between events on a relative basis. As quantification was relative to wild type rather than on an absolute basis, no units are included.
8. Metabolite_F_values: for Table S8. F values for effects of ictB, SBPase (in cases where the model was better with a SBPase effect) and event. These analyses are done for each metabolite included in Table S7, and show effects of the explanatory variables ictB, SBPase, and individual event.
9. Biomass_2020: for Table S9. Biomass and grain yield at harvest, for ictB, ictB/SBPase and wild type sorghum plants in spring 2020. Four ictb/SBPase double construct and four ictB single construct events were included.
10. Biomass_2017: for Table S10. Biomass and grain yield at harvest, in chilled and non-chilled sorghum plants containing the ictB transgene (along with wild type controls) in fall 2017. Four ictB single construct events were included. Chilling treatment involved temperatures of 10 °C day / 7 °C night in growth chambers.

All the variables in the file are explained as below:
o Type (IctB-SBPase and IctB). This refers to whether a plant is wild type, single construct (contains only the ictB transgene) or double construct (contains both the ictB and SBPase transgenes).
o Code: these codes are shorter labels to refer to each transgene event for the sake of convenience.
o Alternate_Code: these codes are shorter labels to refer to each transgene event for the sake of convenience.
o Event Number: these are unique labels for each transgenic events.
o Construct Number: these are labels for each transgenic construct (either the ictB single construct or the ictB/SBPase double construct).
o year (i): this refers to the year in which the study was conducted (2014, 2016, 2017, or 2020)
o transgene or Transgenic: whether the transgene was present
o construct or Type : whether the ictB or the ictB/SBPase construct was present (double, single, wildtype):
o temp: leaf temperature during the measurement
o A: carbon assimilation rate, in μmol m-2 s-1
o gs: stomatal conductance, in mol m-2 s-1
o CI: intercellular carbon dioxide concentration, in parts per million or μL L-1
o fvfm:FV’/FM’ (maximal potential photosystem II quantum yield under light adapted conditions), dimensionless ratio
o phipsill: ΦPSII (maximal potential photosystem II quantum yield under light adapted conditions), dimensionless ratio
o qP: photochemical quenching, i.e. ratio of ΦPSII to FV’/FM’ , dimensionless ratio
o iwue: intrinsic water use efficiency, i.e. ratio of carbon assimilation rate to stomatal conductance, in units of μmol mol-1
o event: individual transgenic / transformation event
o Vmax: substrate-saturated in vitro activity of the SBPase enzyme, in μmol m-2 s-1
o ID: identification number of sample
o ΔCT1: difference in cycle times to threshold during gene expression (quantitative PCR) assay, between ictB and the reference gene GAPDH, in units of cycles
o ΔCT2: cycle times to threshold during gene expression (quantitative PCR) assay, between SBPase and the reference gene GAPDH, in units of cycles
o GAPDH: cycle times to threshold for the reference gene GAPDH (glyceraldehyde phosphate dehydrogenase)
o IctB: cycle times to threshold for the gene of interest ictB
o SBPase: cycle times to threshold for the gene of interest SBPase
o v1 to v267 represent individual metabolite (see the heading immediately above the labels v1, v2, etc.). Variables v268-v272 refer to total (summed) metabolite levels for particular pathways of interest.
o leaf: Leaf and stem dry biomass (in grams)
o seed: Seedhead dry biomass (in grams)
o biomass: Total (leaf, stem + seed head) dry biomass (in grams)
o harvind: ratio of seed head dry biomass to total dry biomass
o treatment (chilled and nonchilled): “Chilled” plants were grown under warm greenhouse conditions (32 °C day / 25 °C night) for 6 or 8 weeks, then switched to chilling temperatures under growth chamber conditions (10 °C / 7 °C night) for 8 days, and were then returned to greenhouse growing conditions.
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