The sequences of all the ribosomal protein-encoding genes of rice (RPL and RPS) were retrieved from RGAP-DB. The sequence identification and validation in RAP-DB, NCBI, and some other databases were carried out as described in Moin et al. (2016a,b) and Saha et al. (2017) to ensure their specificity. Primers were designed specifically for each of the identified genes using primer3 tool for studying their expression in response to different water stress related treatments.

To examine the responses of RP genes to different water stress treatments under greenhouse conditions (32 ± 2°C with relative humidity of 55 ± 5%), 1-month-old rice plants were subjected to limited water and drought stress treatments. The rice seeds of the variety, BPT-5204 were dehulled and surface sterilized with 70% ethanol for 1 min followed by the treatment with 4% sodium hypochlorite twice for 10 min. Seeds were then washed with sterile double-distilled water for 3–5 times, blot-dried and germinated on MS medium in the growth room (28 ± 2°C) for 15 days. After this, they were transferred to pots containing black alluvial soil in the greenhouse where they were further allowed to grow for 1 month. The 1-month-old WT rice plants were subjected to three different watering conditions; one set of plants were provided with ample amounts of water, about 500 ml/d as required for normal growth of rice. The other set was maintained under limited water conditions by providing minimal water (150 ml/day) so as to maintain barely moist conditions in the soil. There was no overlay or additional water in the pots of these plants. The third set of plants were allowed to grow under drought conditions without any water at all. These conditions were maintained up to 3 weeks (21 days). Before the initiation of drought stress treatments, a trial experiment was conducted to check the time that takes for the complete wilting of the majority of rice plants after withholding water from the pots. It was observed that more than 50% of rice plants became wilted between 15 and 21 days. Hence, 21 days treatment was considered as the permanent wilting point (PWP) for investigations on gene expression studies under drought stress. Drought stress induced to plant are usually represented in percent Field Capacity (FC), which mainly depends on the soil type. According to India water portal (http://www.indiawaterportal.org) for alluvial soils available in South India, which has been used in the current study for rice cultivation, the PWP occurs between 10 and 18% FC. In the present experiment, PWP was noticed on 21 day drought response, accordingly the FC on 3, 7, 15, and 21 day was ~60, 40, 20, and 15%, respectively. The leaf and root tissue samples were collected separately after 3, 7, 15, and 21 days treatment from each experimental sample in biological triplicates. The WT plants grown under well-watered conditions were considered as a control to normalize the expression of the corresponding treated samples.

A comprehensive expression analysis of all the RP genes that are involved in the assembly of both large and small subunits of rice (RPL and RPS) was performed using qRT-PCR. Total RNA was extracted from the root and shoot tissue samples separately in three biological replicates. About 100 mg samples of homogenized leaf and root tissues were transferred to sterile, DEPC (Diethyl Pyrocarbonate)-treated Eppendorf tubes containing 1 ml Trizol solution (Sigma-Aldrich, US) and centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant was transferred to a fresh vial and 200 μl of chloroform was added. After a gentle mix and incubation for 5 min, the vial was centrifuged at 12,000 rpm at 4°C for 15 min. This step produced three phases, a red organic phase (containing proteins), an interphase (containing DNA) and a clear aqueous phase that contains RNA. This RNA containing phase was transferred to a fresh tube and 500 μl of ice cold isopropanol was added and allowed to stand for 10 min, after which the vial was centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant was discarded and the clear RNA pellet was washed with 75% ethanol prepared in DEPC-treated water and centrifuged at 7,500 rpm at 4°C for 5 min. The pellet was air dried and dissolved in 20 μl DEPC-treated water. To avoid DNA contamination, total RNA was treated with RNase free DNase1 (Sigma-Aldrich, USA). The quality and quantity of RNA was checked using a spectrophotometer (NanoDrop Technologies Inc., USA) at A260/230 and A260/280 nm wavelengths. All the steps were carried out at 4°C and the vials involved in RNA isolation were treated with DEPC before use.

The cDNA was synthesized using reverse transcriptase (Takara, Clonetech, USA) and diluted in 1:7 proportion. The quantitative real time PCR (qRT-PCR) was performed to analyze the transcript levels of RPL and RPS genes in leaf and root tissues of rice plants grown at different levels of water as per Moin et al. (2016a). Tissues from plants grown with adequate water supply were used as control to normalize the corresponding fold change in gene expression. The qRT-PCR was performed using SYBR Green® Premix (Takara Bio, USA). The qRT-PCR cyclic conditions included an initial denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 15 s, an annealing temperature according to each gene for 25 s and 72°C for 30 s followed by a melting curve. The qRT-PCR was performed as three technical and biological repeats and the fold change was calculated using the ΔΔC_T method (Livak and Schmittgen, 2001).

The full-length cDNA sequence of RPL23A (460 bp) was retrieved from RGAP-DB. The sequence was also validated in RAP-DB and NCBI databases. When the RPL23A sequence from all the databases showed perfect match, the primers were designed according to the sequence with NcoI and XbaI restriction sites at the forward and reverse ends, respectively for subsequent cloning steps. The sequence was amplified from WT rice cDNA. The pRT100 (Addgene, A05521) was used as an intermediate vector to release the 35S promoter and poly-A tail along with the gene using NcoI and XbaI sites. The expression cassette of RPL23A was then cloned into the binary vector, pCAMBIA1300 using PstI digestion. The vector carrying expression cassette of RPL23A was mobilized into Agrobacterium tumefaciens strain, EHA-105 for plant transformation.

The same variety of rice, BPT-5204 (Samba Mahsuri) that has been used in the expression studies was also used in generating the transgenic plants. The binary vector pCAMBIA1300 carrying full-length expression cassette of RPL23A was transformed into rice using Agrobacterium-mediated in planta transformation. The in planta transformation was performed according to the protocol described previously (Moin et al., 2016a). The transformation efficiency of the transgenic plants was almost the same as reported earlier (20%). After infection of the plants with Agrobacterium in the T₀ generation, they were transferred to pots containing alluvial soil and maintained at 30 ± 2°C with 16 h of light followed by 8 h dark photoperiod. The T-DNA of the binary vector carries hptII, as a resistance marker for the selection of transgenic plants in subsequent generations using the antibiotic, Hygromycin.

The seeds obtained from the Agrobacterium-treated plants (T₀) were allowed to germinate on MS selection medium containing the antibiotic, Hygromycin (50 mgl⁻¹). The seedlings that arose from the seed germinated on the selection medium were allowed to grow in the greenhouse and were further confirmed by PCR analysis of various elements present on the T-DNA of the binary vector. The plasmid of pCAMBIA1300 carrying RPL23A expression cassette was used as a Positive Control (PC), whereas the plants that were rescued from non-germinated seeds on selection medium followed by recovery on the selection-free medium were used as Negative Control (NC), and were represented as Null Segregant (NS) plants.

The genomic DNA was isolated using leaf tissue from T₂ generation transgenic plants using the CTAB method with certain modifications. About 150 mg of leaf samples were used for grinding. To this, 1 ml of CTAB buffer + 20 μl β-mercapto-ethanol were added and the macerated tissue was incubated at 65°C. After 1 h, samples were centrifuged at 11,000 rpm for 15 min. To the supernatant that was transferred to a fresh vial, an equal volume of phenol: chloroform: iso-amyl alcohol (25:24:1) was added and the mixture was incubated at 4°C for 5 min followed by centrifugation at 5,000 rpm for 8 min. Incubation at 4°C (instead of the usual incubation at room temperature) resulted in better separation of proteins and nucleic-acids. An equal volume of chloroform: iso-amyl alcohol (24:1) was then added to the supernatant, which was further incubated at 4°C for 15 min with gentle shaking followed by centrifugation at 12,000 rpm for 12 min. This step was repeated twice. The clear upper phase was taken and an equal volume of iso-propanol was added and incubated for 8–12 h at −20°C. Genomic DNA was pelleted down, washed with 70% ethanol, air dried and dissolved in 100 μl nuclease free water. This resulted in the extraction of high-yield (2,000 ng μl⁻¹) and good quality genomic DNA, which was free from protein and salt contamination (260/280 = 1.8, 260/230 = 2.1) and used in Southern analysis without the requirement for further purification.

Southern-blot hybridization was performed to confirm the transgenic nature of plants and also to determine the number of copies of T-DNA integration present in the genomes of the transgenic plants. Southern-blot hybridization was performed using 15 μg of high-quality genomic DNA. The genomic DNA was digested with SphI restriction enzyme, whose site is absent in the T-DNA of the RPL23A overexpression vector and incubated at 37°C overnight. The PCR-amplified, DIG-dUTP (Roche, Germany) labeled fragment of the Hygromycin resistance gene (hptII) was used as a probe in Southern hybridization analysis. Since RPL23A, which was used for overexpression studies in the present report is endogenous to rice, hptII was used as a probe instead of RPL23A. This was to avoid the multiple banding patterns that might arise with the use of endogenous RPL23A (which also has gene copies) even in wild-type rice. The probe binding was detected with anti-DIG-alkaline phosphatase enzyme and NBT/BCIP substrate.

Total RNA was isolated from the leaves and roots of 1-month-old RPL23A transgenic and NS plants using Trizol (Sigma-Aldrich, US) method as described earlier. The first strand cDNA was synthesized from 2 μg of total RNA using reverse transcriptase (Takara Bio, Clontech, USA). About 2 μl of 1:7 diluted cDNA was used for analyzing the RPL23A gene transcript levels in the selected transgenic lines. The rice RPL23A specific primers, designed through primer-3 tool were used in semi-Q and qRT-PCR. The cycle conditions for semi-Q PCR included an initial denaturation at 94°C for 3 min, followed by 26–28 repeated cycles of the 94°C for 30 s, 55°C for 25 s and 72°C for 30 s. This was followed by a final extension for 5 min at 72°C. The rice Actin, act1 and tubulin, β-tub were used as endogenous control genes in qRT-PCR analyses for normalization.

Semi-Q and qRT-PCR analyses were performed on T₂ generation rice transgenic plants to separate low and high expression lines of RPL23A transgenic rice plants. In the semi-Q analysis, the transcript levels of RPL23A in transgenic lines was measured based on the band intensity observed on the 1.5% agarose gel compared with the NS. The transgenic lines with weak and increased band intensity compared with the band intensities of NC were categorized as low and high expression lines, respectively. The transcript levels in transgenic lines were further validated through qRT-PCR. The same cDNA that was diluted in 1:7 proportions used in semi-Q PCR was also used in qRT-PCR to analyze the transcript levels in two classes of RPL23A lines (low and high) that were separated through semi-Q PCR. The cDNA synthesized from null or non-germinated seedlings was used as a Negative Control or Null Segregant (NC or NS) to normalize the expression pattern in qRT-PCR. The qRT-PCR reaction was performed using SYBR master mix (Takara Bio, USA) and the reaction conditions were similar as described above with an annealing temperature specific to RPL23A gene (56°C). The qRT-PCR data were analyzed according to the ΔΔC_T method (Livak and Schmittgen, 2001).

In our previous report, we have already shown that the enhanced expression of RPL23A by the integrated 35S tetrameric enhancers in one stable Ds line among the activation tagged rice population resulted in enhanced WUE (Moin et al., 2016a). In the present study, we have overexpressed RPL23A and generated independent transgenic plants to validate the previous findings on the enhancement of WUE in rice by the activated expression of RPL23A. The two high expression lines along with NS were screened for WUE. After selection on Hygromycin (50 mg l⁻¹) medium, the transgenic plants along with NS were transferred to black alluvial soil in the pots and provided ample amounts of water (up to 500 ml/day) for the first 4 weeks of transfer. After this, the overlaid water was removed from the pots and watering was restricted to about 150 ml/day so that only moist conditions were maintained in the soil. Limited water treatments were given to the rice plants as described earlier (Moin et al., 2016a). The phenotypic observations on the confirmed RPL23A transgenic plants in the T₃ generation were performed in comparison with NS. The various phenotypic characters measured included the total number of tillers, productive tillers (tillers with panicles), panicle length and plant height. The data of all phenotypic parameters were collected from five individual plants of each transgenic line and NS.

Atmospheric Carbon exist as two isotopes, ¹³C and ¹²C with a molar ratio of 1:99. The diffusion of Carbon through stomata and assimilation by Ribulose-1,5-bis carboxylase/ oxygenase (RuBisCo) during photosynthesis discriminates between the two isotopes. During limited water supply, stomatal aperture tends to close to reduce water loss through transpiration resulting in a decrease in the concentration of intercellular CO₂ (Ci). This discrimination by RuBisCo between the Carbon isotopes is high when internal CO₂ is high and decreases with a decrease in Ci. Thus, the Δ¹³C value, which is the relative ratio of ¹³C/¹²C, expressed relative to the PDB standard, of a plant tissue reflects the capacity of a plant for gaseous exchange through stomata, integral Ci and overall WUE of a plant (Martin and Thorstenson, 1988; Farquhar et al., 1989; Bassett, 2013). The Δ¹³C is inversely related to WUE; lesser the Δ¹³C, higher will be WUE. The Δ¹³C value was measured using 500 mg of mature leaf samples collected from NS and four RPL23A T₁ generation transgenic plants (parental plants, which were subsequently confirmed by Southern-blot analysis) after 1-month of growth under limited water conditions. Samples were dried at 65°C for 3 day in a hot-air oven, finely powdered and carbon isotope ratios were analyzed using an Isotope Ratio Mass Spectrometer (IRMS).

Chlorophyll fluorescence is a measure of the activity of photosystem II (PSII). It is also an indicator of plant response to environmental stresses and has been used to assess the overall photosynthetic performance of a plant (Murchie and Lawson, 2013). The chlorophyll fluorescence of RPL23A transgenic plants in the T₂ generation was measured using a portable instrument, MINI-PAM essentially according to the manufacturer's protocol (Walz, Effeltrich, Germany; Murchie and Lawson, 2013; Batra et al., 2014). Readings were taken in the two high expression lines, just before the treatment (1 month after transfer to pots) and 1 month after the start of limited water conditions. Each set of readings was taken in triplicates and the quantum efficiency (F_v/F_m) of transgenic plants was compared with the NS plants and represented in the form of bar diagrams.

The two high expression lines of RPL23A were selected to check the transcript levels of other RP genes to gain insights into whether they are differentially regulated by RPL23A. For this qRT-PCR was performed using primers of 70 RP genes in two selected high expression lines. The fold change was calculated using ΔΔC_T and the expression was normalized with an NS plant that was used in the separation of high and low expression lines.

After 7 day of germination, the transgenic and NS seedlings were transferred to test tubes. To evaluate the response of RPL23A transgenic lines to NaCl and simulated drought stress (other than WUE), the T₃ generation seedlings were allowed to grow in the solutions of NaCl (100 mM) and PEG-6000 (10%) prepared in distilled water for 10 day. The corresponding seedlings grown in water (without a stress-inducing agent) were considered as untreated or control. The fresh weight (FW) and root lengths (RL) of two high expression lines were measured using a scale bar after 10 day of treatment with PEG and NaCl with respect to NS plants. The FW and RL were measured in triplicates and the data were represented in the form of bar diagrams.

The osmoprotectant, proline was estimated in two high expression lines and corresponding NS plants at the T₃ generation seedling stage. The two high expression and NS seedlings, after an initial germination for 1 week were allowed to grow in salt (100 mM), PEG (10%) and water for 10 days. About 100 mg of leaf-derived tissue from 10 day old treated and untreated seedlings was homogenized in 5 ml of 3% aqueous sulfosalicylic acid. The leaf homogenate was then centrifuged at 12,000 rpm for 15 min. Then, 400 μl of supernatant was mixed with equal volumes of 400 μl acid ninhydrin and glacial acetic acid and incubated for 1 h at 100°C. The reaction mixture was then mixed with 800 μl toluene. The organic phase was used for measuring absorbance at 520 nm wavelength using toluene as a blank. Proline concentration was measured from the standard curve using the method described by Bates et al. (1973).

The chlorophyll content was also measured in the seedling stage of T₃ generation transgenic and NS plants. The chlorophyll was extracted using 100 mg of leaf tissues in 80% acetone and absorption of the extracts was measured at OD 663 nm and 645 nm using a UV spectrophotometer (UV-1800 Shimadzu) as per the established protocols (Arnon, 1949; Zhang et al., 2009). The samples used for chlorophyll estimation included leaves of 10 day old transgenic and NS seedlings that were grown in NaCl (100 mM), PEG (10%) and water collected in triplicates to measure the concentrations of Chl-a, Chl-b, and total chlorophyll (Chl-t).

The transcript levels of six stress-specific genes, such as bZIP23, WRKY72, DREB2B, LEA3-1, SNAC1, and SNAC2 were studied in high expression lines of RPL23A. These genes were selected as they have been reported to be to be involved in conferring tolerance to different abiotic stresses, particularly drought and salt in rice. This analysis was performed in the shoot and root tissues of PEG-treated transgenic seedlings for 48 h, and the expression data was normalized with corresponding untreated transgenic samples.

The qRT-PCR, phenotypic observations (tillering and seed yield) and physiological experiments (fresh weight, root length and proline and chlorophyll contents) were carried out in three biological and three technical replicates. The fold change in each qRT-PCR experiment in the present study is normalized with two rice specific reference genes, act1 and β-tub. The mean fold change obtained after normalization with these two genes was considered as final fold change. The mean of the observations (qRT-PCR, phenotypic and physiological studies) was represented in the form of bar diagrams constructed using SigmaPlot v11. One-way ANOVA was used to study the statistical significance, and the significance was represented at P < 0.05 with asterisks in the graphs. The qRT-PCR data was also represented in the form of heat maps constructed using the mean of fold change obtained from biological and technical triplicates. Heat maps were developed using Morpheus program.

A keyword search of “ribosomal” resulted in the identification of 70 representative genes that are involved in the assembly of both large and small subunits (Moin et al., 2016a,b; Saha et al., 2017).

To gain insights into whether RP genes respond differentially to dehydration treatments at the greenhouse level, WT rice plants were subjected to different dehydration treatments and the expression profiles of both large and small subunit genes were analyzed periodically. After 21 days treatment, rice plants grown under drought conditions became completely dried, whereas those grown under limited water started to wilt (Supplementary Figure 1). The fold change obtained from dehydration treated samples was compared with corresponding control samples grown under normal conditions providing ample amounts of water.

All the RP genes exhibited differential expression in response to water deficit treatments in both shoot and root tissues. The majority of genes became upregulated at some time point or the other indicating that the majority of them respond to dehydration treatments positively. The genes that exhibited ≥2-fold transcript level on the log₂ scale were considered as upregulated. In shoots, about 30 RPL genes (88%) became activated under limited water conditions after 3 days of treatment. Among these, 25 (73%), 12 (35%), and 11 (32%) RPL genes, respectively maintained the expression levels consistently up to 7, 15, and 21 days. In roots, initially 11 RPL genes (32%) became upregulated after the onset of limited water stress and as the treatment progressed, many other genes began to express. After 21 days, 25 RPL genes (65%) showed significant expression. About 75–80% of RPS genes became upregulated during 3–15 day treatment, of which some of them were downregulated as the treatment progressed. About 60% maintained continuously enhanced expression levels up to the last time period (21 days) in shoots. However, only 7 RPS genes were activated in roots on the 3rd day of treatment, but as the treatment progressed, 18 (52%), 27 (79%), and 29 (85%) genes became upregulated. Under drought conditions, 83% of RPL genes started expression in shoots and as the treatment continued, some of them became down-regulated. Around 76, 52, and 41% of RPL genes, respectively expressed at 17, 15, and 21 day time intervals. In roots, the number of RPL genes that expressed gradually increased from 44% on 3rd day to 52% on 7th day, 64% on 15 day, and 70% on the 21st day. The number of RPS genes expressed in shoots under drought stress ranged from 35 to 88%, while in roots, the number of upregulated RPS genes ranged from 20 to 80%.

To analyze the level of expression of 70 RP genes in detail, they were categorized as low, if the fold is 2–5; moderate, if they exhibit 5–10 fold; and significant, if the transcript levels were more than 10-fold. Among the genes that showed significant of expression in shoots at a given time period under both limited water and drought treatments included RPL6, 7, 19, 21.2, 23A, 18, 26, 27, 28, 36, 37, and 51; and RPS4, 5, 7a, 10, 17, 18a, 19, 20, 21, and 23a, whereas, RPL6, 7, 10, 11, 13a, 13b, 18p, 21, 23A, 24, 26, 37, and 44; and RPS9, 10, 18, 21, 23, 24, 25, and 27 became significantly upregulated in roots in response to both the treatments.

In shoots, RPL6, 7, 19-3, 21.2, 18A, 23A, 24, 31, 34, 35A, 37.1, and 51; RPS4, 10, 18, 19, 23, 24, 26, and 29 showed high expression (>10-fold) throughout the duration of dehydration treatments, in both limited water and drought conditions at all the time intervals (3, 7, 15, and 21 day) studied. Similarly, in roots, RPL6, 7, 11, 13b, 18p, 23A, 24, 26.1, 30, 31, 32, 35a, 36.2; RPS4, 6, 6a, 9, 10, 13, 17, 18, 23, 25, 27, 28, and 29 exhibited high levels of transcripts after the onset of the treatment until the last time point. RPL6, 7, 23A, 24, and 31; RPS4, 10 and 18 became upregulated in both shoot and root tissues (Supplementary Figures 2A–P). These genes can be considered to be promising in bringing about tolerance under water deficit treatments as they consistently became upregulated in both the tissues. This also clearly indicates that they have a common role in inducing tolerance to both water deficit and drought stress conditions. The overlap in up-regulation (Supplementary Figure 3A) and down-regulation (Supplementary Figure 3B) of RP genes in response to limited water and drought conditions in the shoot and root tissues separately were represented in the form of Venn diagrams. Supplementary Table 1 provides a detailed list of genes that exhibited overlap in the up-regulation (Supplementary Tables 1A–H) and down-regulation (Supplementary Tables 1I–P) in the shoot and root tissues at each time point.

The upregulation of these RPL genes is in accordance with our previous observations, where we have studied the expression of RPL genes in treatments with stress-inducing agents and at different developmental stages of the cultivar BPT-5204, which is also the test material in the present study (Moin et al., 2016b). The expression patterns of RPL (Figure 1) and RPS (Figure 2) genes have been represented in the form of heat maps, which were generated using the mean of fold change obtained from different biological and technical triplicates. The dark-colored grids in the heat maps indicate significantly enhanced expression, while light-colored grids represent weak expression.

The RPL23A construct (Figure 3A) was confirmed by restriction digestion using PstI enzyme, which released the RPL23A expression cassette of ~1,200 bp (Figure 3B). The binary vector was also confirmed by PCR analysis. The seeds obtained from the primary generation Agrobacterium-treated plants (T₀) were screened on Hygromycin selection medium (50 mg l⁻¹) as the binary vector contains hptII as a selection marker that confers resistance to the antibiotic, Hygromycin. The putative transgenic seedlings continued further growth within 4–5 day after inoculation, while non-transgenic and WT seeds became bleached. After selection, plants were analyzed by PCR amplification using hptII gene in the T-DNA using specific primers (Figure 3C). Since the WT and NS plants also contain RPL23A in their genome, hptII was used for PCR analysis, but not RPL23A. About 400 seeds were infected with Agrobacterium carrying the binary vector, pCAMBIA-RPL23A, from these 95 (23%) were found to be positive through antibiotic selection and PCR amplification. The hptII resistant and PCR positive plants obtained from selfing of the Agrobacterium-treated plants were selected for developing homozygous lines and Southern-blot hybridization. Further, among the positive transgenic plants, only those that were confirmed through Southern-blot hybridization were selected for detailed molecular investigations.

Some of the transgenic plants that were positive for antibiotic selection and PCR amplification were selected for Southern-blot hybridization analysis. The independent nature of T-DNA integration into the genome of transgenic plants was identified by the different restriction fragments of the transgenic plants binding to the probe. Of the nine samples analyzed, single insertions were found in five lines. Of these five lines, three were high expression and two were low expression lines. Although there were many high and low expression lines observed through semi-Q and qRT-PCR, only those that were confirmed through Southern-blot hybridization were selected for further molecular investigations.

The T₂ generation rice transgenic plants were separated into low and high expression lines based on the band intensity of RPL23A transcripts observed through semi-Q PCR on an agarose gel. Rice actin was used to normalize the expression patterns (Figure 4A). Based on the band intensity, lines were categorized into low and high expression using primers specific to RPL23A. A total of ten transgenic plants were selected for expression analysis through semi-Q PCR. The transcript levels of some of them were further determined by qRT-PCR using the NS as a negative control to normalize the expression pattern of other low and high expression lines. The lines with transcript levels <5-fold were considered as low, while those with more than 5-fold increase in expression were categorized as high expression lines. Three lines, L23A-15.8.1, L23A-12.6.22, and L23A-21.3.3 were identified as high expression lines having transcript levels ranging from 25 to 30-fold in shoots (Figure 4B) and up to 45-fold in roots (Figure 4C). The two transgenic lines, L23A-5.23.1 and L23A-1.2.11 that were identified as low expression lines through semi-Q PCR were selected to compare their expression with high expression lines and it was observed that the transcript levels of L23A-5.23.1 and L23A-1.2.11 ranged between 2- and 5 fold in both shoots and roots corroborating the observations made through semi-Q PCR. The high expression lines, L23A-15.8.1 and L23A-12.6.22 with single gene insertion observed through Southern analysis were selected for all the physiological and quantitative studies and their experimental readings were compared with the NS plants.

To check whether overexpression of RPL23A is associated with differential expression of other members of RP gene family, we have studied the expression levels of all the RP genes in two high expression lines of RPL23A. About 50% of both RPL and RPS family members showed an expression of more than 2-fold increase. RPL29, 30, 31, 32, 35, 37, and 38, and RPS4, 10, 17, 18a, 24, and 25 became significantly upregulated indicating the possibility of a cross talk between RPL23A and these genes (Figure 5).

The high expression lines of RPL23A showed increased yield-related parameters, such as tillering, panicle number and size and total seed yield under limited water conditions with respect to WT (Supplementary Table 2). The Δ¹³C measured in two high, L23A-15.8 and L23A-12.6, and two low expression, L23A-5.23, L23A-1.2 parental lines had values of 19.24, 19.14, 21.5, and 20.9‰, respectively with respect to NS, which had 23.75‰ (Figure 6A). The chlorophyll fluorescence of RPL23A transgenic plants in the T₂ generation was measured using MINI-PAM. The two high expression lines, L23A-15.8.1 and L23A-12.6.22 showed a quantum efficiency (Fv/Fm) of 0.86 and 0.88, respectively just before the water withdrawal treatments, with respect to NS (0.82). Interestingly, 1 month after limited water availability (150 ml/day), both the lines had the quantum efficiency of 0.80, which was close to the well-watered conditions and significantly greater than the NS, whose quantum efficiency was only 0.65 (Figure 6B).

The transgenic seedlings exhibited better tolerance to simulated drought (10% of PEG-6000) and salt (100 mM NaCl) treatments (Supplementary Figure 4). The root length (RL) of NC and transgenic seedlings was 4 and 6.5, respectively under normal (untreated) conditions. In response to PEG, there was not much change in RL. However, the RL under NaCl treatment of untreated seedlings increased from 4 to 6 cm, which is equivalent to that of transgenic seedlings (Figure 6C). The untreated seedlings of high expression lines exhibited a total FW of 155–160 mg, which was 1.5 fold higher than the corresponding NS seedlings (100 mg). Under PEG, the high expression lines had an FW of 75–80 mg, while NC exhibited only 45 mg. Similarly, the FW of high expression lines in response to NaCl treatment was up to 100 mg, which is 2-fold greater than NS (50 mg) (Figure 6D).

The proline content was found to be increased in both transgenic and NS seedlings after treatment with PEG and NaCl. However, the proline in transgenic seedlings was 0.5–1 fold higher than NS (Figure 6E). The increase in proline content indicates that cytosolic osmotic potential is maintained in transgenic lines even under the conditions of stress.

The content of Chl-a in seedlings of NS and high expression lines, L23A-15.8.1 and L23A-12.6.22 under untreated condition was up to 9.5, 15.8, and 15.3 mg/g, respectively. After exposure to PEG and NaCl treatments, the level was slightly reduced to 13.04 and 13.7 mg/g in high expression lines but higher than NC (6.73 mg/g) (Figure 6F). The Chl-b content in high expression lines was around 3.5 mg/g under normal condition with respect to NS (2.02 mg/g), while in response to PEG and NaCl, Chl-b was slightly decreased but higher than NS (Figure 6G). The total chlorophyll content under normal conditions in high expression lines was around 17 mg/g compared with NC (11.5 mg/g). After treatment with NaCl and PEG, the Chl-t in high expression lines was in the range of 11–14 mg/g, which was significantly higher than NS under both the conditions (Figure 6H).

Overexpression studies of LEA3-1, bZIP23, NAC, and DREB genes in rice showed enhanced drought tolerance and yield (Chen et al., 2008; Xiang et al., 2008; Zheng et al., 2009; Liu et al., 2014). The transcription factors, such as OsbZIP23, OsNAC1, OsNAC2, and OsWRKY72 have also been shown to be highly expressed by drought and salt stresses in rice. All these genes also became upregulated in the high expression lines of RPL23A under simulated drought treatment (Supplementary Figure 5). Particularly, the genes bZIP23, DREB2B, LEA3-1, and SNAC-1 became several fold upregulated in the transgenic rice plants overexpressing RPL23A suggesting that it played an important role in the proper targeting of these stress-specific proteins under the conditions of stress.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.