**3. Results**

### *3.1. Phenotypes of Swarna and DTY-IL under Reproductive Stage Drought Stress*

In the 2014 and 2015 dry season (DS) drought-stress field trials at IRRI, DTY-IL and Swarna, showed similar grain yield (Figure 1A) and plant height (Figure 1B) under irrigated conditions. Under reproductive-stage drought stress height was reduced in both genotypes to a similar degree (Figure 1B). While yield was reduced in both lines under drought, DTY-IL achieved about double the average grain yield compared to Swarna (Figure 1A). In addition, three weeks after draining the field, Swarna showed clear signs of leaf rolling. Leaf rolling was also observed for Swarna in the greenhouse experiment. While there was no visible di fference in flag-leaf morphology between both genotypes under the well-watered condition (Figure 1C), a prominent leaf rolling phenotype in Swarna was observed after ten days of drought stress with complete leaf-rolling on the 12th day of drought stress (Figure 1D).

**Figure 1.** The effect of reproductive stage drought on yield, height and flag-leaf morphology. The average grain yield ( **A**) and plant height (**B**) across the 2014 and 2015 dry season (DS) field trial of DTY-Il and Swarna under irrigated and drought condition ( *N* = 2–9). Flag-leaf phenotypes of the DTY-IL and Swarna under well-watered conditions ( **C**) and the leaf-rolling phenotype during drought-stress ( **D**) under controlled greenhouse conditions. The asterisks (\*) indicates a significant di fference (Student's *t* test, \* *p* < 0.01).

### *3.2. Generating a Transcriptional Map of the Moderate RDS Response in Rice*

Mapping rates ranged from 77.7–92.9% for flag-leaf and 87.9–92.8% for panicle (Table S1), covering 28,283 and 33,698 genes, respectively. Sample clustering and heatmap visualization of log2-transformed, normalized count data demonstrated clear separation between genotypes and treatments for both flag-leaf and panicle samples (Figures S2A,B and S3A,B). Principal components (PC) analysis showed that the first and second PC explained 93% of the total variation for flag-leaf (Figure S2C) and 81% for panicle tissue (Figure S3C). Biological replicates of each genotype-treatment combination clustered together and the treatment effect was greater than the genotype effect for both tissues (Figures S2C and S3C). An exception was a single Swarna panicle sample, which was removed from all further analyses as an outlier (Figure S4). The sum of the non-redundant/unique log2 normalized genes with significant changes of expression at FDR adjusted *p*-value ≤ 0.05 across four different contrasts were 17,616 and 18,614 for flag-leaf and panicle tissue, respectively. Pairwise DE analysis for all genotype-treatment combinations identified DEGs of significance for flag-leaf and panicle (Figure S5), and a Venn diagram was used to visualize three categories of unique and common responses in flag-leaf (Figure 2A and Figure S6A,B, Table 1) and panicle (Figure 2B and Figure S6C,D, Table 1).

**Figure 2.** Gene di fferential expression and identification. Venn diagram of di fferentially expressed

genes (DEGs) for flag-leaf (**A**) and panicle (**B**) tissues in Swarna and DTY-IL under reproductive drought stress (RDS) at a false discovery rate (FDR) adjusted *p*-value < 0.05 and −1 ≤ log2-ratio ≤ +1 (fold change ≥ 2 and ≤ 0.5). The three highlighted boxes for each tissue represent the common DEGs (red), unique to Swarna (yellow), and unique to DTY-IL (blue). SWAC = Swarna control, SWAD = Swarna under RDS, ILC = DTY-IL control, ILD = DTY-IL under RDS.


**Table 1.** A summary of GO enrichment analysis of differentially expressed genes (DEGs) common to both genotypes, and unique to DTY-IL and Swarna for flag-leaf and panicle tissues under RDS.

### 3.2.1. Expression Profiles of Drought-Responsive Genes in the Flag-Leaf Tissue under RDS

A total of 4180 genes were found to be drought-responsive in flag-leaves of both Swarna and DTY-IL (Figure 2A, Table 1). Gene ontology (GO) enrichment analysis showed functional enrichment among upregulated DEG for transcription, regulation of biological processes, and oxidation-reduction (Table S2-1), while the shared downregulated DEGs were mainly associated with transmembrane transport, localization, and post-translational protein modification (Table S2-2).

In Swarna flag-leaves a total of 515 (188 up- and 327 downregulated) genes were uniquely drought-responsive (Table 1). While no significant GO terms were detected for upregulated DEGs (Table 1), significant GO terms for uniquely downregulated DEGs were largely related to post-translational protein modification, photosynthesis, defense response, and programmed cell death (Figure S7A). Pathway enrichment suggested photosynthesis, ubiquinone, and other terpenoid-quinone biosynthesis, as well as glutathione-mediated detoxification II and tyrosine biosynthesis (Table S3), to be significantly downregulated. MapMan visualization supported the downregulation of cell wall, carbon metabolism, secondary metabolism, and light reaction in Swarna flag-leaves (Figure 3A and Figure S9A).

In DTY-IL flag-leaves 108 (74 up- and 34 downregulated) DEGs were uniquely drought-responsive. While no significant GO terms could be associated with downregulated DEGs, oxidation-reduction, response to stress, and response to stimulus were among the significant GO terms in upregulated DEGs (Figure S7B). Pathway enrichment suggested phenylpropanoid biosynthesis, dhurrin, xylan, and scopoletin biosynthesis, as well as detoxification of reactive carbonyls in chloroplasts to be uniquely upregulated under RDS (Table S3). This was supported by MapMan visualization, showing upregulation of cell wall, lipids, and secondary metabolism (Figure 3B and Figure S9B).

Numerous overrepresented *cis*-elements were found in the group of 327 promoters of uniquely downregulated DEGs in Swarna in flag-leaf, which are mostly involved as binding sites for dehydration responsive genes. These include ACGTATERD1, IBOX, PREATPRODH, MYCATERD1, MYCATRD22, CCAATBOX1, and MYB2AT (Table S4-1). The overrepresented motifs in a set of 74 promoters in the uniquely upregulated DEGs in IL in flag-leaf were mostly functioning upon induction of dehydration stress through the ACGTATERD1 motif (Table S4-2).

3.2.2. Expression Profiles of Drought-Responsive Genes in the Panicle Tissue under RDS

A total of 4799 genes were found to be drought-responsive in panicles of both Swarna and DTY-IL (Figure 2B; Table 1). Enriched GO categories of the upregulated DEGs related to post-translational protein modification and response to stress (Table S5-1), while enriched GO terms of the downregulated DEGs were mostly related to transmembrane transport, carbohydrate metabolic process, and localization (Table S5-2).

In Swarna panicles, a total of 487 (184 up and 303 downregulated) genes were found uniquely drought-responsive (Table 1). No significant GO terms were identified within the uniquely upregulated DEGs. For the uniquely downregulated genes, significant GO terms included oxidation-reduction as well as monooxygenase activity, tetrapyrrole, and heme-binding (Figure S8A). Significantly enriched pathways associated with downregulated DEGs were related to DNA replication, diterpenoid biosynthesis, phenylpropanoid biosynthesis, and photosynthesis (Table S6). MapMan visualization supported the downregulation of cell wall, lipids, secondary metabolism, amino acids, as well as carbohydrate metabolism (Figure 3C and Figure S9C).

In DTY-IL panicle 164 (108 up and 56 downregulated) DEGs were uniquely drought-responsive (Table 1). No significant GO enrichment was identified among the uniquely downregulated DEGs of DTY-IL. Prevalent GO terms of upregulated DEGs were related to protein amino acid phosphorylation as well as oxidation-reduction and carbohydrate metabolic processes (Figure S8B). The most significantly enriched pathways in the panicle tissue of DTY-IL upregulated DEGs were related to propanoate metabolism, methylerythritol phosphate pathway, diterpenoid biosynthesis, camalexin biosynthesis, and circadian rhythm in plants (Table S6). This was supported by MapMan visualization, showing upregulation of cell wall, lipids, secondary metabolism as well as amino acids (Figure 3D and Figure S9D).

Overrepresented *cis*-acting elements in the group of 303 promoters of the uniquely downregulated DEGs in Swarna in panicle tissue mostly involved in dehydration response like the ACGTATERD1, MYBCOREATCYB1, ABRELATEDRD1, and IBOX motifs (Table S7-1). Overrepresented *cis*-elements in a set of 108 promoters of uniquely upregulated DEGs in DTY-IL in panicle tissue were mostly related to dehydration response like the MYB2AT, MYBCOREATCYCB1, and ACGTABOX (Table S7-2).

### *3.3. Drought-Stress-Related Gene Modules within the Transcriptional Map*

WGCNA identified 21 distinct co-expressed modules with di fferent expression patterns in flag-leaf (designated as FL-M1 to FL-M21, capturing 17,616 genes) (Figure S10B,C), and 23 distinct modules for panicle network (designated as P-M1 to P-M23, capturing 18,614 genes) (Figure S10E,F).

More than 70% of genes were distributed in the FL-M1 and FL-M2, and P-M1 and P-M2 (common response between DTY-IL and Swarna under RDS and control) for flag-leaf and panicle networks, respectively (Table S8-1,S8-2; Figure S11), signifying a common response shared between Swarna and DTY-IL. Modules FL-M1 and FL-M2 in the flag-leaf network showed "localization" and "transport" as the most enriched GO terms (Table S9-1). In the panicle network, P-M1 genes were enriched for functions related to "RNA processing" while the P-M2 module was linked with "localization" and "transport" (Table S9-2).
