Rothamsted Repository Download

G-protein signaling components have been attributed many biological roles in plants but the extent of involvement of G-protein coupled receptor 1 ( GCR1 ) with the G α ( GPA1 ) remained 19 unknown. To address this, we have performed transcriptomic analyses on Arabidopsis gpa1- 20 5gcr1-5 double mutant and identified 656 differentially expressed genes (DEGs). MapMan and 21 Gene Ontology analyses revealed global transcriptional changes associated with external 22 stimulus, cell wall organization/biogenesis and secondary metabolite process among others. 23 Comparative transcriptomic analyses using the single and double mutants of gcr1-5 and gpa1-5 24 identified 194, 139 and 391 exclusive DEGs respectively, whereas 64 DEGs were common to all 25 three mutants. Further, pair wise comparison of DEGs of double mutant with single mutants of 26 gcr1-5 or gpa1-5 showed about one-third and over half common DEGs, respectively. Further 27 analysis of the DEGs exclusive to the double mutant using protein-protein interaction networks 28 revealed molecular complexes associated with nitrate and light signaling and plant-pathogen 29 interactions among others. Physiological and molecular validation of nitrate-response revealed 30 the sensitivity of germination to low N in the double mutant, and differential expression of 31 nitrate transporter and nitrate reductase in all three mutants. Taken together, GCR1 and GPA1 32 work in partnership as well as independently to regulate different pathways.

other than putative and unspecified ones (Fig. 5B). While many of the MYB family members were found to be down-regulated in the double mutant, none of the transcription factors of AP2-139 EREB and WRKY families were down-regulated. On the other hand, in the bHLH and C2H2   Secondary metabolism. The GO class associated with secondary metabolites were found to be 195 an important category, so we checked the involvement of GCR1/GPA1 in regulating the genes of 196 secondary metabolism. We found that 107 DEGs belong to the biosynthesis of flavonoids and isoprenoids based on Mapman as well as pathway analysis using AraCyc database (Fig. S4, Table 2). The genes involved in flavonoid biosynthesis include 2-oxoglutarate, dihydroflavanol-  Fig. 3. Flavonoid biosynthesis was also found to be regulated in 204 our previous studies using single mutants of GPA1 and GCR1, but many more genes belonging 205 to this category are differentially regulated in the double mutant. Thus, we found that out of the 206 11 genes that regulate flavonoid biosynthesis in the double mutant, only 2 genes were found to 207 be regulated in both the single mutants, whereas 5 genes were regulated in gpa1-5 and 3 genes 208 were regulated in gcr1-5.

209
Development. We also detected the association of 80 DEGs in developmental processes (Fig. 4). involved in developmental processes and hence, shows a larger convergence with 17 genes being 217 common between them. Only four DEGs were found to be common to gcr1-5 16 in this category. 218 We confirmed the basic trends of regulation in the mutant in this category using qPCR on the up-219 regulated (AT2G35710 and AT1G78860) as well as down-regulated (VSP2 and AT2G02160) 220 genes (Fig. 3).  were detected in sub-cluster 1, 2, 3 and 4, respectively. All seven sub-clusters details are 287 mentioned in Table S7. The sub-cluster 1 includes transcriptional regulators associated with light (Long Hypocotyl in Farred1) (Fig. 8, Table S7). The sub-clusters 4 also include transcription three hours for the next three days (72 h) and total % seeds germinated and time taken for 50% 305 germination were used to compare WT and mutants. All of them started germinating around 30 h 306 after soaking and seeds of both the single mutants and wild type were broadly similar at all 307 nitrate doses, both in terms of total germination at 72 h (95-100%) and the time taken for 50% 308 germination ( Fig. 9). However, the double mutant was sensitive to low nitrate level (12.5 mM) 309 on both counts. It had significantly lower level of total germination (80%) and also significantly 310 slower germination rate, as the time taken for 50% seeds to germinate was delayed by 4 h 311 relative to the WT (Fig. 9B).

313
In order to investigate whether these mutants are affected in genes encoding nitrate uptake and  Our results also show for the first time that gcr1 mutant shows altered dose-dependent   We have provided the first evidence against such exclusive approach using parallel functional 342 genomic analyses of mutants of Arabidopsis GCR1 16 /GPA1 17 from a gene discovery perspective. 343 We showed there by venn selection that 30% of all GCR1-responsive genes and 57% of all 344 GCR1-regulated processes were similar to those of GPA1, though there were also many that did  have been verified by qRT-PCR (Fig. 3) and a larger list of the top 10 DEGs is given in Table 1.

361
Functional annotation and MapMan pathway enrichment analysis showed that these DEGs were 362 involved many pathways such as response to external stimulus, primary and secondary cell wall 363 modulation/biosynthetic processes, plant immunity, secondary metabolism, nitrogen signaling 364 and light signaling among others.

365
The genes/processes identically regulated in all 3 mutants can be best explained by GCR1 indicates that the effects of GCR1 mutation are carried over to the double mutant but GPA1 387 mutation has no effect on these genes, either in the gpa1-5 mutant or in the double mutant. The 388 best explanation for this is that GCR1 regulates these genes through some other partner, which 389 may be another GPA alike isoform that is yet to be identified, or the Gβ and/or Gγ, RGS, XLG 390 components of heterotrimeric G-protein complex, or through a totally different, non-G-protein 391 signalling mechanism. While testing these possibilities is beyond the scope of the current study, hydrogen peroxide metabolic and catabolic processes in the gpa1-5 mutant and response to 407 starvation, phosphate starvation and nutrient levels in the gcr1-5 mutant (Fig. 7A). This clearly 408 suggests that modulation of cell wall composition requires both GCR1 and GPA1 function. the double mutant as compare to either of the single mutants (Fig 7B).
analysis. Log2fold change value of 1.0 and p-value of 0.05 was used as a cut-off for differential-

519
The amplifications were carried out using biological triplicates, two of which were the same as 520 those used for microarray. Serial dilutions were used to check for primer efficiency and only 521 those primers that worked at 100 ± 10% efficiency were used for all qPCR analyses. The 522 specificity of primer pairs was confirmed by melting curve analysis of the amplicons. Actin2   For qPCR analyses, surface sterilized and stratified seeds of the wild type and all three mutants 541 were grown in B5 medium containing 12.5 and 30 mM KNO 3 at 22 ᵒC ± 1 in a growth chamber.

542
Root tissues (~100 mg) were used to extract their total RNA using Trizol (Invitrogen, USA) as