The essential role of sugar metabolism in the acclimation response of Arabidopsis thaliana to high light intensities

Summary Analyses of mutants impaired in assimilate export from chloroplasts revealed that carbohydrates as primary output of photosynthesis control expression of nuclear genes associated with plastidial processes such as acclimation to high light intensities.


Conditions MgProtoIX Lhcb2 a vs b a vs c a vs d b vs c b vs d c vs d a vs b a vs c a vs d b vs c b vs d c vs d
HL LL t 30min t 60min t 240min t 480min Document S1 (Table 4). Statistical analysis (ANOVA/Tukey-Kramer) of contents of redox components of wild-type and mutant plants in a time series after transfer from LL conditions (i.e. a PFD of 30 µmol·m -2 ·s -1 ) to HL (i.e. a PFD of 300 µmol·m -2 ·s -1 ) within 4h compared to HL grown plants. The biotypes are denoted, a = Col-0; b = tpt-2, c = adg1-1, d = adg1-1/tpt-2. Significance levels of P < 0.05 or P < 0.01 are indicated by light or dark blue colours.

Compound Conditions
Document S1 (Table 5). Statistical analysis (ANOVA/Tukey-Kramer) of metabolite contents of wild-type and mutant plants in a time series after transfer from LL conditions (i.e. a PFD of 30 µmol·m -2 ·s -1 ) to HL (i.e. a PFD of 300 µmol·m -2 ·s -1 ) within 4h and 48h compared to HL-or LLgrown plants. The conditions are denoted, a = t 0 (LL); b = t 4h (HL), c = t 48h (HL), d = HL. Significance levels of P < 0.05 or P < 0.01 are indicated by light or dark blue colours.

Sucrose, D-(8TMS)
Glucose The starch-free adg1-1 single mutant contained the highest number of significantly altered genes in the static comparison, particularly under LL-conditions (Fig. 6A) Table S3A). These data suggest an elevated input into lipid formation and/or secondary metabolism as a consequence of a deficiency in the nightpath of photoassimilate export from the chloroplast.
The functional pattern of genes changed appreciably after 48h in HL (Fig. 6, Table 1,   Supplementary Table S4A). There were a total of 209 genes specifically altered in adg1-1 compared to the wild type. Interestingly, although the category `lipid metabolism´ was missing among the 58 highly up-regulated genes, there were seven genes belonging to this category among the 151 highly down-regulated genes, with four of these genes related to `lipid degradation´. Hence, the transient enhancement of lipid synthesis (i.e. 4h after LL/HLtransfer) was replaced by an inhibition of lipid degradation in the long term. Furthermore five genes involved in `major CHO metabolism´, 12 genes related to `proteins´ (including seven genes connected to `protein synthesis´), as well as six genes related to `regulation of transcription (RT)´ were highly up-regulated (Table 1). Moreover, the up-regulation of genes involved in `protein synthesis´ was accompanied by a down-regulation of 13 genes related to `protein degradation´, again suggesting an enhanced production and/or maintenance of proteins. Furthermore 21, 11, and seven genes related to `RT´, `stress´ and `signalling´, respectively, were highly down-regulated in adg1-1 48h after LL/HL-transfer (Supplementary   Table S4A). Moreover, the gene coding for the glucose 6-phosphate/phosphate translocator 2 (GPT2; At1g61800) was highly up-regulated in adg1-1.

(B) tpt-2 vs Col-0
Under LL-conditions, a limitation in the day-path of photoassimilate export from the chloroplast in the tpt-2 mutant resulted only in the down-regulation of a single gene encoding a disulfide isomerase-like protein (AtPDIL 5-4, At4g27080; Supplementary Table S2B).
The number of highly altered genes in tpt-2 was increased to 36, 4h after the plants were transferred to HL. There were only two genes down-regulated, amongst them again AtPDIL 5-4 and a protein of unknown function (At4g27080; Supplementary Table S3B). The 34 highly up-regulated genes comprised three genes related to `major carbohydrate metabolism´ and seven, significantly over-represented genes involved in `protein degradation´ (Table 1). After 48h in HL, again, there were more genes highly up-regulated (104) than down-regulated (3) ( Supplementary Table S4B). Interestingly, AtPDIL 5-4 still belonged to the down-regulated genes. Within the group of up-regulated genes there were three significantly over-represented functional clusters, i.e. `cell wall´, `hormone metabolism´ and `stress´ (Table 1). Furthermore, genes related to `RT´ and `development´ were represented with at least five members. Ten genes, including a MAPkinase (At1g01560), were connected to `signalling´, in particular, `calcium signalling´ (nine genes).

(c) adg1-1/tpt-2 vs Col-0
Despite the relative high number of specifically altered genes in the adg1-1 mutant, surprisingly, the combined deficiency in the day-and night-path of photoassimilate export resulted in an appreciable lower number of differentially regulated genes in adg1-1/tpt-2.
Under LL-conditions there were only 21 genes highly up-or down-regulated in the double mutant (Supplementary Table S2C). Among the nine up-regulated genes in adg1-1/tpt-2, remarkably, there were three genes related to `abiotic stress´, sub-category heat. All three genes belong to the putative HSP20-type protein of unknown function (At1g53540, At3g46230, At5g12020). Of the 12 down-regulated genes one half is related to `histone proteins´ and `chromatin structure´, suggesting that parts of the DNA was not associated with proteins and/or the plants contained less DNA. The only gene dramatically downregulated in the overlapping area of adg1-1/tpt-2 and tpt-2 was, as expected, the TPT gene (Supplementary Table S2D). In the overlapping region between adg1-1/tpt-2 and adg1-1 there were 13 up-and 33 down-regulated genes found. Among the down-regulated genes there were six genes related to `chromatin structure´ as well as five genes involved in `protein degradation´, again suggesting a function of starch and/or soluble sugars in protein maintenance and chromatin structure.
Both genes were more pronounced up-regulated in adg1-1/tpt-2 compared to adg1-1 or tpt-2. Amongst the down-regulated genes there was a bHLH-type transcription factor (At4g17880; MYC4), which was highly and specifically down-regulated with a log2-ratio of -5.17 in adg1-1/tpt-2. Strikingly the same gene was also highly down-regulated after 48h in HL (log2-ratio = -4.21) and even in LL (log2-ratio = -3.68). A closer inspection of the expression profiles (eFP browser, Winter et al., 2007) revealed that this gene is highly regulated by various stress conditions, like for instance oxidative stress, and it responds to jasmonate (Fernández-Calvo et al., 2011). Moreover, the presence of externally fed Suc induces the expression of At4g17880. Furthermore, MYC4 has been identified to be one of the key players in the regulation of glucosinolate biosynthesis (Schweizer et al., 2013). At t 48h there were eight more transcriptional regulators within the group of 112 highly downregulated genes in adg1-1/tpt-2. Moreover, the functional categories `amino acids´, `cell wall´, and `major carbohydrate metabolism´ were significantly over-represented (Table 1).
Interestingly, among the 36 highly up-regulated genes, 22 were plastome-encoded and belonged to the categories `PS light reaction´ (16 genes), `protein biosynthesis´ (five genes) and `lipid metabolism´ (one gene). Moreover, there were four nuclear-encoded genes involved in `RT´ up-regulated in adg1-1/tpt-2.

Supplementary Document S3
Genes associated with `major carbohydrate´-and `lipid metabolism´ as well as `transport´ were commonly up-regulated 4h after LL/HL-transfer The group of commonly regulated genes as a response to LL/HL-transfer comprised also metabolic genes. Strikingly, seven and 11 genes associated with `lipid-´ and `major CHO metabolism´, respectively, were up-regulated only transiently at t 4h vs t 0 (Supplementary Table S7A).
As an analysis with ATTED-II (version 6.1) revealed, all genes apart from a plastidial thioesterase (At1g54570) belong to a regulatory network (Supplementary Fig. S3B; Supplementary Table S7A).
Moreover, in the category `lipid metabolism´ there were ten and seven genes downregulated at t 4h vs t 0 and t 48h vs t 0 , respectively, with an overlap of five genes. The ten downregulated genes at t 4h vs t 0 form, with the exception of At5g08030, a large network ( Supplementary Fig. S3C), whereas there is no evidence for any exceptional network formation with the seven down-regulated genes at t 48h vs t 0 (not shown).
disproportionating enzyme 2 [At2g40840; Lu & Sharkey, 2004] and glucan posphorylase 2 [At3g46970]) were highly up-regulated 4h after LL/HL-transfer in both wild-type and mutant plants. Again this regulation of genes involved in carbohydrate metabolism also occurred in the starch-free background (i.e. adg1-1 and adg1-1/tpt-2). Interestingly, three of the starchrelated genes belong to the co-expression network of phospholipase A (At2g06925; Supplementary Table S7A). Moreover, within the co-regulation network of genes belonging to the category `major CHO metabolism´, there was a chloroplast localised AMP activated protein kinase induced 4h and 48h after transfer to HL (Supplementary Table S7, A and B).
The respective gene (At5g39790) appears to contain a starch binding domain (SUBA3 database; Heazlewood et al., 2007;Tanz et al., 2013) and might hence be involved in carbohydrate metabolism or signalling. Most strikingly, the expression of `starch related´ genes occurred independently from the presence of starch (i.e. in the starch-free background adg1-1), suggesting that the resulting proteins might have additional unknown functions.
In the category `secondary metabolism´ there were five and 11 genes up-regulated, related to `flavonoids´ in a broader sense at t 4h vs t 0 and t 48h vs t 0 , respectively (Supplementary Table S7, A and B). Only three of these genes were commonly differently regulated at both time points. In addition, three more up-regulated genes belonged to the sub-category `isoprenoids´ and `miscellaneous´.
Genes associated with specific `transport processes´ were differentially expressed most pronounced 4h after LL/HL-transfer Genes associated with `transport processes´ were de-regulated both at 4h and 48h after LL/HL-transfer. Of the 14 up-regulated genes at t 4h vs t 0 , only two genes were also found at t 48h vs t 0 . Likewise, of the 18 down-regulated transport associated genes at t 4h vs t 0 , only six were also found at t 48h vs t 0 (Supplementary  et al., 2007), strongly responds to elevated soluble sugar levels (Kunz et al., 2010, Schmitz et al., 2012, e.g. in starch-free mutants or after feeding of exogenous sugars to the plants (Heinrichs et al., 2012). GPT2 was highly up-regulated with log2-ratios between 3.7 and 6.7 at t 4h vs t 0 in all plant lines