Physiological and structural traits contribute to thermotolerance in wild Australian cotton species.

BACKGROUND AND AIMS: Five species of cotton (Gossypium) were exposed to 38°C days during early vegetative development. Commercial cotton (Gossypium hirsutum) was contrasted with four wild cotton species (G. australe, G. bickii, G. robinsonii and G. sturtianum) that are endemic to central and northern Australia. METHODS: Plants were grown at daytime maxima of 30°C or 38°C for 25 d, commencing at the four-leaf stage. Leaf areas and shoot biomass were used to calculate relative rates of growth and specific leaf areas. Leaf gas exchange measurements revealed assimilation and transpiration rates, as well as electron transport rates (ETR) and carboxylation efficiency (CE) in steady-state conditions. Finally, leaf morphological traits (mean leaf area and leaf shape were quantified), along with leaf surface decorations, imaged using scanning electron microscopy. KEY RESULTS: Shoot morphology was differentially affected by heat, with three of the four wild species growing faster at 38°C than at 30°C, whereas early growth in G. hirsutum was severely inhibited by heat. Areas of individual leaves and leaf numbers both contributed to these contrasting growth responses, with fewer, smaller leaves at 38°C in G. hirsutum. CO2 assimilation and transpiration rates of G. hirsutum were also dramatically reduced by heat. Cultivated cotton failed to achieve evaporative cooling, contrasting with the transpiration-driven cooling in the wild species. Heat substantially reduced ETR and CE in G. hirsutum, with much smaller effects in the wild species. We speculate that leaf shape, as assessed by invaginations of leaf margins, and leaf size contributed to heat dispersal differentially among the five species. Similarly, reflectance of light radiation was also highly distinctive for each species. CONCLUSIONS: These four wild Australian relatives of cotton have adapted to hot days that are inhibitory to commercial cotton, deploying a range of physiological and structural adaptations to achieve accelerated growth at 38°C.

Leaf phosphorus fractions vary with leaf economic traits among 35 Australian woody species.

Adaptations of plants to phosphorus (P) deficiency include reduced investment of leaf P in storage (orthophosphates in vacuoles), nucleic acids and membrane lipids. Yet, it is unclear how these adaptations are associated with plant ecological strategies. Five leaf P fractions (orthophosphate P, Pi ; metabolite P, PM ; nucleic acid P, PN ; lipid P, PL ; and residual P, PR ) were analysed alongside leaf economic traits among 35 Australian woody species from three habitats: one a high-P basalt-derived soil and two low-P sandstone-derived soils, one undisturbed and one disturbed by human activities with artificial P inputs. Species at the undisturbed low-P site generally exhibited lower concentrations of total leaf P ([Ptotal ]), primarily associated with lower concentrations of Pi , and PN . The relative allocation of P to each fraction varied little among sites, except that higher PL per [Ptotal ] (rPL ) was recorded at the undisturbed low-P site than at the high-P site. This higher rPL , reflecting relative allocation to membranes, was primarily associated with lower concentrations of leaf nitrogen at the undisturbed low-P site than at the high-P site. Associations between leaf P fractions and leaf nitrogen may provide a basis for understanding the variation in plant ecological strategies dependent on soil P availability.

A universal protocol for high-quality DNA and RNA isolation from diverse plant species.

Next-generation sequencing demands high-quality nucleic acid, yet isolating DNA and RNA is often challenging, particularly from plant tissues. Despite advances in developing various kits and reagents, these products are tailored to isolation of nucleic acid from model plant tissues. Here we introduce a universal lysis buffer to separate nucleic acid from various plant species, including recalcitrant plants, to facilitate molecular analyses, such as quantitative PCR (qPCR), transcriptomics, and whole-genome sequencing (WGS). The protocol is a modification of the original CTAB methods, which leads to nucleic acid isolation from many plant species, including monocots and eudicots. The lysis buffer consists of hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), Tris base, ethylenediaminetetraacetic acid (EDTA) and ß-mercaptoethanol (ßME). The modified CTAB method enables the isolation of nucleic acid from small amounts of plant tissues (e.g., 15-100 mg) in a timely manner, which is well-suited for a large number of samples and also when adequate sample collection is a limiting factor. The protocol isolates not only DNA from various plant species but also RNA. This makes it highly effective for molecular analyses compared to previously described CTAB methods optimised for DNA isolation. The appropriate concentration of the components enables high-quality DNA and RNA isolation from plant tissues simultaneously. Additionally, this protocol is compatible with commercially available columns. For DNA and RNA to be qualified for next-generation sequencing platforms, the protocol is supplemented with columns to purify either DNA or RNA from the same tissue to meet high standards for sequencing analyses. This protocol provides an ideal approach to overcome potential obstacles in isolating high-quality DNA or RNA from a wide range of plant species for downstream molecular analysis.

Pre-Treatment of Rice Plants with ABA Makes Them More Tolerant to Multiple Abiotic Stress.

Multiple abiotic stress is known as a type of environmental unfavourable condition maximizing the yield and growth gap of crops compared with the optimal condition in both natural and cultivated environments. Rice is the world's most important staple food, and its production is limited the most by environmental unfavourable conditions. In this study, we investigated the pre-treatment of abscisic acid (ABA) on the tolerance of the IAC1131 rice genotype to multiple abiotic stress after a 4-day exposure to combined drought, salt and extreme temperature treatments. A total of 3285 proteins were identified and quantified across the four treatment groups, consisting of control and stressed plants with and without pre-treatment with ABA, with 1633 of those proteins found to be differentially abundant between groups. Compared with the control condition, pre-treatment with the ABA hormone significantly mitigated the leaf damage against combined abiotic stress at the proteome level. Furthermore, the application of exogenous ABA did not affect the proteome profile of the control plants remarkably, while the results were different in stress-exposed plants by a greater number of proteins changed in abundance, especially those which were increased. Taken together, these results suggest that exogenous ABA has a potential priming effect for enhancing the rice seedlings' tolerance against combined abiotic stress, mainly by affecting stress-responsive mechanisms dependent on ABA signalling pathways in plants.

Unique and Shared Proteome Responses of Rice Plants (Oryza sativa) to Individual Abiotic Stresses.

Food safety of staple crops such as rice is of global concern and is at the top of the policy agenda worldwide. Abiotic stresses are one of the main limitations to optimizing yields for sustainability, food security and food safety. We analyzed proteome changes in Oryza sativa cv. Nipponbare in response to five adverse abiotic treatments, including three levels of drought (mild, moderate, and severe), soil salinization, and non-optimal temperatures. All treatments had modest, negative effects on plant growth, enabling us to identify proteins that were common to all stresses, or unique to one. More than 75% of the total of differentially abundant proteins in response to abiotic stresses were specific to individual stresses, while fewer than 5% of stress-induced proteins were shared across all abiotic constraints. Stress-specific and non-specific stress-responsive proteins identified were categorized in terms of core biological processes, molecular functions, and cellular localization.

Proteomic analysis of the meristematic root zone in contrasting genotypes reveals new insights in drought tolerance in rice.

Drought is responsible for major losses in rice production. Root tips contain meristematic and elongation zones that play major roles in determination of root traits and adaptive strategies to drought. In this study we analysed two contrasting genotypes of rice: IR64, a lowland, drought-susceptible, and shallow-rooting genotype; and Azucena, an upland, drought-tolerant, and deep-rooting genotype. Samples were collected of root tips of plants grown under control and water deficit stress conditions. Quantitative proteomics analysis resulted in the identification of 7294 proteins from the root tips of IR64 and 6307 proteins from Azucena. Data are available via ProteomeXchange with identifier PXD033343. Using a Partial Least Square Discriminant Analysis on 4170 differentially abundant proteins, 1138 statistically significant proteins across genotypes and conditions were detected. Twenty two enriched biological processes showing contrasting patterns between two genotypes in response to stress were detected through gene ontology enrichment analysis. This included identification of novel proteins involved in root elongation with specific expression patterns in Azucena, including four Expansins and seven Class III Peroxidases. We also detected an antioxidant network and a metallo-sulfur cluster assembly machinery in Azucena, with roles in reactive oxygen species and iron homeostasis, and positive effects on root cell cycle, growth and elongation.

The first long-read nuclear genome assembly of Oryza australiensis, a wild rice from northern Australia.

Oryza australiensis is a wild rice native to monsoonal northern Australia. The International Oryza Map Alignment Project emphasises its significance as the sole representative of the EE genome clade. Assembly of the O. australiensis genome has previously been challenging due to its high Long Terminal Repeat (LTR) retrotransposon (RT) content. Oxford Nanopore long reads were combined with Illumina short reads to generate a high-quality ~ 858 Mbp genome assembly within 850 contigs with 46× long read coverage. Reference-guided scaffolding increased genome contiguity, placing 88.2% of contigs into 12 pseudomolecules. After alignment to the Oryza sativa cv. Nipponbare genome, we observed several structural variations. PacBio Iso-Seq data were generated for five distinct tissues to improve the functional annotation of 34,587 protein-coding genes and 42,329 transcripts. We also report SNV numbers for three additional O. australiensis genotypes based on Illumina re-sequencing. Although genetic similarity reflected geographical separation, the density of SNVs also correlated with our previous report on variations in salinity tolerance. This genome re-confirms the genetic remoteness of the O. australiensis lineage within the O. officinalis genome complex. Assembly of a high-quality genome for O. australiensis provides an important resource for the discovery of critical genes involved in development and stress tolerance.

Multiple Abiotic Stresses Applied Simultaneously Elicit Distinct Responses in Two Contrasting Rice Cultivars.

Rice crops are often subject to multiple abiotic stresses simultaneously in both natural and cultivated environments, resulting in yield reductions beyond those expected from single stress. We report physiological changes after a 4 day exposure to combined drought, salt and extreme temperature treatments, following a 2 day salinity pre-treatment in two rice genotypes-Nipponbare (a paddy rice) and IAC1131 (an upland landrace). Stomata closed after two days of combined stresses, causing intercellular CO2 concentrations and assimilation rates to diminish rapidly. Abscisic acid (ABA) levels increased at least five-fold but did not differ significantly between the genotypes. Tandem Mass Tag isotopic labelling quantitative proteomics revealed 6215 reproducibly identified proteins in mature leaves across the two genotypes and three time points (0, 2 and 4 days of stress). Of these, 987 were differentially expressed due to stress (cf. control plants), including 41 proteins that changed significantly in abundance in all stressed plants. Heat shock proteins, late embryogenesis abundant proteins and photosynthesis-related proteins were consistently responsive to stress in both Nipponbare and IAC1131. Remarkably, even after 2 days of stress there were almost six times fewer proteins differentially expressed in IAC1131 than Nipponbare. This contrast in the translational response to multiple stresses is consistent with the known tolerance of IAC1131 to dryland conditions.

Photosynthetic traits of Australian wild rice (Oryza australiensis) confer tolerance to extreme daytime temperatures.

KEY MESSAGE: A wild relative of rice from the Australian savannah was compared with cultivated rice, revealing thermotolerance in growth and photosynthetic processes and a more robust carbon economy in extreme heat. Above ~ 32 °C, impaired photosynthesis compromises the productivity of rice. We compared leaf tissues from heat-tolerant wild rice (Oryza australiensis) with temperate-adapted O. sativa after sustained exposure to heat, as well as diurnal heat shock. Leaf elongation and shoot biomass in O. australiensis were unimpaired at 45 °C, and soluble sugar concentrations trebled during 10 h of a 45 °C shock treatment. By contrast, 45 °C slowed growth strongly in O. sativa. Chloroplastic CO2 concentrations eliminated CO2 supply to chloroplasts as the basis of differential heat tolerance. This directed our attention to carboxylation and the abundance of the heat-sensitive chaperone Rubisco activase (Rca) in each species. Surprisingly, O. australiensis leaves at 45 °C had 50% less Rca per unit Rubisco, even though CO2 assimilation was faster than at 30 °C. By contrast, Rca per unit Rubisco doubled in O. sativa at 45 °C while CO2 assimilation was slower, reflecting its inferior Rca thermostability. Plants grown at 45 °C were simultaneously exposed to 700 ppm CO2 to enhance the CO2 supply to Rubisco. Growth at 45 °C responded to CO2 enrichment in O. australiensis but not O. sativa, reflecting more robust carboxylation capacity and thermal tolerance in the wild rice relative.

Genome survey sequencing of wild cotton (Gossypium robinsonii) reveals insights into proteomic responses of pollen to extreme heat.

Heat stress specifically affects fertility by impairing pollen viability but cotton wild relatives successfully reproduce in hot savannas where they evolved. An Australian arid-zone cotton (Gossypium robinsonii) was exposed to heat events during pollen development then mature pollen was subjected to deep proteomic analysis using 57 023 predicted genes from a genomic database we assembled for the same species. Three stages of pollen development, including tetrads (TEs), uninucleate microspores (UNs) and binucleate microspores (BNs) were exposed to 36°C or 40°C for 5 days and the resulting mature pollen was collected at anthesis (p-TE, p-UN and p-BN, respectively). Using the sequential windowed acquisition of all theoretical mass spectra proteomic analysis, 2704 proteins were identified and quantified across all pollen samples analysed. Proteins predominantly decreased in abundance at all stages in response to heat, particularly after exposure of TEs to 40°C. Functional enrichment analyses demonstrated that extreme heat increased the abundance of proteins that contributed to increased messenger RNA splicing via spliceosome, initiation of cytoplasmic translation and protein refolding in p-TE40. However, other functional categories that contributed to intercellular transport were inhibited in p-TE40, linked potentially to Rab proteins. We ascribe the resilience of reproductive processes in G. robinsonii at temperatures up to 40°C, relative to commercial cotton, to a targeted reduction in protein transport.

Comparisons of photosynthetic and anatomical traits between wild and domesticated cotton.

Mesophyll conductance (gm) is a crucial leaf trait contributing to the photosynthetic rate (AN). Plant domestication typically leads to an enhancement of AN that is often associated with profound anatomical modifications, but it is unclear which of these structural alterations influence gm. We analyzed the implication of domestication on leaf anatomy and its effect on gm in 26 wild and 31 domesticated cotton genotypes (Gossypium sp.) grown under field conditions. We found that domesticated genotypes had higher AN but similar gm to wild genotypes. Consistent with this, domestication did not translate into significant differences in the fraction of mesophyll occupied by intercellular air spaces (fias) or mesophyll and chloroplast surface area exposed to intercellular air space (Sm/S and Sc/S, respectively). However, leaves of domesticated genotypes were significantly thicker, with larger but fewer mesophyll cells with thinner cell walls. Moreover, domesticated genotypes had higher cell wall conductance (gcw) but smaller cytoplasmic conductance (gcyt) than wild genotypes. It appears that domestication in cotton has not generally led to significant improvement in gm, in part because their thinner mesophyll cell walls (increasing gcw) compensate for their lower gcyt, itself due to larger distance between plasmalemma and chloroplast envelopes.

Patterns of gene expression in pollen of cotton (Gossypium hirsutum) indicate downregulation as a feature of thermotolerance.

Reproductive performance in plants is impaired as maximum temperatures consistently approach 40°C. However, the timing of heatwaves critically affects their impact. We studied the molecular responses during pollen maturation in cotton to investigate the vulnerability to high temperature. Tetrads (TEs), uninucleate and binucleate microspores, and mature pollen were subjected to SWATH-MS and RNA-seq analyses after exposure to 38/28°C (day/night) for 5 days. The results indicated that molecular signatures were downregulated progressively in response to heat during pollen development. This was even more evident in leaves, where three-quarters of differentially changed proteins decreased in abundance during heat. Functional analysis showed that translation of genes increased in TEs after exposure to heat; however, the reverse pattern was observed in mature pollen and leaves. For example, proteins involved in transport were highly abundant in TEs whereas in later stages of pollen formation and leaves, heat suppressed synthesis of proteins involved in cell-to-cell communication. Moreover, a large number of heat shock proteins were identified in heat-affected TEs, but these proteins were less abundant in mature pollen and leaves. We speculate that the sensitivity of TE cells to heat is related to high rates of translation targeted to pathways that might not be essential for thermotolerance. Molecular signatures during stages of pollen development after heatwaves could provide markers for future genetic improvement.

The phosphoproteome of rice leaves responds to water and nitrogen supply.

The scarcity of freshwater is an increasing concern in flood-irrigated rice, whilst excessive use of nitrogen fertilizers is costly and contributes to environmental pollution. To co-ordinate growth adaptation under prolonged exposure to limited water or excess nitrogen supply, plants employ complex systems for signalling and regulation of metabolic processes. There is limited information on the involvement of one of the most important post-translational modifications (PTMs), protein phosphorylation, in plant adaptation to long-term changes in resource supply. Oryza sativa cv. Nipponbare was grown under two regimes of nitrogen from the time of germination to final harvest. Twenty-five days after germination, water was withheld from half the pots in each nitrogen treatment and low water supply continued for an additional 26 days, while the remaining pots were well watered. Leaves from all four groups of plants were harvested after 51 days in order to test whether phosphorylation of leaf proteins responded to prior abiotic stress events. The dominant impact of these resources is exerted in leaves, where PTMs have been predicted to occur. Proteins were extracted and phosphopeptides were analysed by nanoLC-MS/MS analysis, coupled with label-free quantitation. Water and nitrogen regimes triggered extensive changes in phosphorylation of proteins involved in membrane transport, such as the aquaporin OsPIP2-6, a water channel protein. Our study reveals phosphorylation of several peptides belonging to proteins involved in RNA-processing and carbohydrate metabolism, suggesting that phosphorylation events regulate the signalling cascades that are required to optimize plant response to resource supply.

Drought by CO2 interactions in trees: a test of the water savings mechanism.

Elevated atmospheric CO2 (eCa ) may benefit plants during drought by reducing stomatal conductance (gs ) but any 'water savings effect' could be neutralized by concurrent stimulation of leaf area. We investigated whether eCa enhanced water savings, thereby ameliorating the impact of drought on carbon and water relations in trees. We report leaf-level gas exchange and whole-plant and soil water relations during a short-term dry-down in two Eucalyptus species with contrasting drought tolerance. Plants had previously been established for 9 to 11 months in steady-state conditions of ambient atmospheric CO2 (aCa ) and eCa , with half of each treatment group exposed to sustained drought for 5 to 7 months. The lower stomatal conductance under eCa did not lead to soil moisture savings during the dry-down due to the counteractive effect of increased whole-plant leaf area. Nonetheless, eCa -grown plants maintained higher photosynthetic rates and leaf water potentials, making them less stressed during the dry-down, despite being larger. These effects were more pronounced in the xeric species than the mesic species, and in previously water-stressed plants. Our findings indicate that eCa may enhance plant performance during drought despite a lack of soil water savings, especially in species with more conservative growth and water-use strategies.

Pollen development in cotton (Gossypium hirsutum) is highly sensitive to heat exposure during the tetrad stage.

The development of gametes in plants is acutely susceptible to heatwaves as brief as a few days, adversely affecting pollen maturation and reproductive success. Pollen in cotton (Gossypium hirsutum) was differentially affected when tetrad and binucleate stages were exposed to heat, revealing new insights into the interaction between heat and pollen development. Squares were tagged and exposed to 36/25°C (day/night, moderate heat) or 40/30°C (day/night, extreme heat) for 5 days. Mature pollen grains and leaves were collected for physiological and proteomic responses. While photosynthetic competence was not compromised even at 40°C, leaf tissues became leakier. In contrast, pollen grains were markedly smaller after the tetrad stage was exposed to 40°C and boll production was reduced by 65%. Sugar levels in pollen grains were elevated after exposure to heat, eliminating carbohydrate deficits as a likely cause of poor reproductive capacity. Proteomic analysis of pure pollen samples revealed a particularly high abundance of 70-kDa heat shock (Hsp70s) and cytoskeletal proteins. While short-term bursts of heat had a minor impact on leaves, male gametophyte development was profoundly damaged. Cotton acclimates to maxima of 36°C at both the vegetative and reproductive stages but 5-days exposure to 40°C significantly impairs reproductive development.

Wild and Cultivated Species of Rice Have Distinctive Proteomic Responses to Drought.

Drought often compromises yield in non-irrigated crops such as rainfed rice, imperiling the communities that depend upon it as a primary food source. In this study, two cultivated species (Oryza sativa cv. Nipponbare and Oryza glaberrima cv. CG14) and an endemic, perennial Australian wild species (Oryza australiensis) were grown in soil at 40% field capacity for 7 d (drought). The hypothesis was that the natural tolerance of O. australiensis to erratic water supply would be reflected in a unique proteomic profile. Leaves from droughted plants and well-watered controls were harvested for label-free quantitative shotgun proteomics. Physiological and gene ontology analysis confirmed that O. australiensis responded uniquely to drought, with superior leaf water status and enhanced levels of photosynthetic proteins. Distinctive patterns of protein accumulation in drought were observed across the O. australiensis proteome. Photosynthetic and stress-response proteins were more abundant in drought-affected O. glaberrima than O. sativa, and were further enriched in O. australiensis. In contrast, the level of accumulation of photosynthetic proteins decreased when O. sativa underwent drought, while a narrower range of stress-responsive proteins showed increased levels of accumulation. Distinctive proteomic profiles and the accumulated levels of individual proteins with specific functions in response to drought in O. australiensis indicate the importance of this species as a source of stress tolerance genes.

Proteomic Responses to Drought Vary Widely Among Eight Diverse Genotypes of Rice (Oryza sativa).

Rice is a critically important food source but yields worldwide are vulnerable to periods of drought. We exposed eight genotypes of upland and lowland rice (Oryza sativa L. ssp. japonica and indica) to drought stress at the late vegetative stage, and harvested leaves for label-free shotgun proteomics. Gene ontology analysis was used to identify common drought-responsive proteins in vegetative tissues, and leaf proteins that are unique to individual genotypes, suggesting diversity in the metabolic responses to drought. Eight proteins were found to be induced in response to drought stress in all eight genotypes. A total of 213 proteins were identified in a single genotype, 83 of which were increased in abundance in response to drought stress. In total, 10 of these 83 proteins were of a largely uncharacterized function, making them candidates for functional analysis and potential biomarkers for drought tolerance.

Salt-Treated Roots of Oryza australiensis Seedlings are Enriched with Proteins Involved in Energetics and Transport.

Salinity is a major constraint on rice productivity worldwide. However, mechanisms of salt tolerance in wild rice relatives are unknown. Root microsomal proteins are extracted from two Oryza australiensis accessions contrasting in salt tolerance. Whole roots of 2-week-old seedlings are treated with 80 mM NaCl for 30 days to induce salt stress. Proteins are quantified by tandem mass tags (TMT) and triple-stage Mass Spectrometry. More than 200 differentially expressed proteins between the salt-treated and control samples in the two accessions (p-value <0.05) are found. Gene Ontology (GO) analysis shows that proteins categorized as "metabolic process," "transport," and "transmembrane transporter" are highly responsive to salt treatment. In particular, mitochondrial ATPases and SNARE proteins are more abundant in roots of the salt-tolerant accession and responded strongly when roots are exposed to salinity. mRNA quantification validated the elevated protein abundances of a monosaccharide transporter and an antiporter observed in the salt-tolerant genotype. The importance of the upregulated monosaccharide transporter and a VAMP-like protein by measuring salinity responses of two yeast knockout mutants for genes homologous to those encoding these proteins in rice are confirmed. Potential new mechanisms of salt tolerance in rice, with implications for breeding of elite cultivars are also discussed.

Proteomes of Leaf-Growing Zones in Rice Genotypes with Contrasting Drought Tolerance.

Plants require a distinctive cohort of enzymes to coordinate cell division and expansion. Proteomic analysis now enables interrogation of immature leaf bases where these processes occur. Hence, proteins in tissues sampled from leaves of a drought-tolerant rice (IAC1131) are investigated to provide insights into the effect of soil drying on gene expression relative to the drought-sensitive genotype Nipponbare. Shoot growth zones are dissected to estimate the proportion of dividing cells and extract protein for subsequent tandem mass tags quantitative proteomic analysis. Gene ontology annotations of differentially expressed proteins provide insights into responses of Nipponbare and IAC1131 to drought. Soil drying does not affect the percentage of mitotic cells in IAC1131. More than 800 proteins across most functional categories increase in drought (and decrease on rewatering) in IAC1131, including proteins involved in "organizing the meristem" and "new cell formation". On the other hand, the percentage of dividing cells in Nipponbare is severely impaired during drought and fewer than 200 proteins respond in abundance when growing zones undergo a drying cycle. Remarkably, the proteomes of the growing zones of each genotype respond in a highly distinctive manner, reflecting their contrasting drought tolerance even at the earliest stages of leaf development.

Label-free and isobaric tandem mass tag (TMT) multiplexed quantitative proteomic data of two contrasting rice cultivars exposed to drought stress and recovery.

Two rice cultivars, IAC1131 (drought tolerant) and Nipponbare (drought sensitive), with contrasting genetic backgrounds and levels of tolerance to drought, were analysed using both label-free and tandem mass tags (TMTs) quantitative proteomics approaches, aiming to elucidate the mechanisms of drought tolerance. Four-week-old seedlings of both cultivars were grown in large soil volumes in the glasshouse under controlled conditions and then exposed to moderate and extreme drought for 7 days, followed by 3 days of re-watering period. Mature leaves were harvested from plants of each treatment for protein extraction and subsequent complementary shotgun proteomic analyses. The data from this study are related to the research article "Quantitative proteomic analysis of two different rice varieties reveals that drought tolerance is correlated with reduced abundance of photosynthetic machinery and increased abundance of ClpD1 protease" (Wu et al., 2016) [1].