Plausible Photosynthetic Parameters that Contribute to Differences in Grain Protein Content among Rice Genotypes

doi:10.21203/rs.3.rs-3215732/v1

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Research Article

Plausible Photosynthetic Parameters that Contribute to Differences in Grain Protein Content among Rice Genotypes

https://doi.org/10.21203/rs.3.rs-3215732/v1

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Aim

Attempts to increase grain protein content (GPC) most often resulted in a reduction of yield in cereals. This trade-off between GPC and yield rises mainly because of shared source of reductants for carbon and nitrogen assimilation. The major intent of this study was to understand this trade-off between GPC and grain yield in rice.

Methods

Based on a previous study, we identified two genotypes contrasting GPC with comparable yield and photosynthesis. We articulated the interrelation between carbon and nitrogen assimilation to understand the phenomenon that led to higher GPC without compromising its yield among the contrasts. We also looked at how these genotypes behaved to ambient and low light intensities in terms of photosynthesis and protein synthesis under limiting electron source.

Results

Our findings suggested that under ambient light, the high GPC genotype has adopted the triose phosphate utilization limitation as a traffic switch to limit the flow of electrons to carbon assimilation and divert the excess electrons to other sinks. The diversion of extra electrons particularly to nitrite reduction is boosted by increased substrates from higher uptake, transport and metabolism of nitrogen in leaf. In low GPC genotype under ambient light intensity excess electrons were quenched in the form of heat.

Conclusions

These findings suggest that efficiency in utilization of electrons by adopting a switch called TPU limitation coupled with better remobilization efficiency can be a promising genotype for breeders to develop a high yielding variety complemented with high GPC.

Grain protein content

Nitrogen transporters

Photosynthesis

Rice

Yield

Rice (Oryza sativa L.) is the staple food for more than half of the world's population, and it is of great importance in many Asian countries, including India (Maclean et al, 2013). Grain protein content is an important factor in rice quality, as it affects not just the nutritional value but also the taste and texture of rice (Hamaker, 1993). In India, where malnutrition is still a significant problem, improving the grain protein content of rice is highly indicative. Poor protein quality has an adverse effect on health, especially during sensitive periods of pregnancy and the first 1000 days of life where, stunting, poor development, and increased risk of later life diseases develop (Martorell, 2017). After the Green revolution, adequate food grains are supplied to people through the public distribution system, and nutritional security should be the next priority. It can be accomplished by improving cereal protein quality. In this regard, improving rice grain protein content and yield are two major targets of rice breeding programmes. However, improving these two traits simultaneously is a difficult task due to negative genetic relationship between them (Munier-Jolain and Salon, 2005). A major setback in achieving this goal is high yielding varieties have reduced protein content (Gu et al., 2015) and grain yield decreases when grain protein content (GPC) is enhanced in a variety for inexplicable reasons. According to Zhu et al. (2018), rice varieties grown in higher levels of CO₂ experienced a notable decrease in their protein content, with an average reduction of 10.3% compared to rice varieties grown in ambient CO₂ conditions. This negative relationship illustrates the trade-off between carbon and nitrogen metabolism. Though different strategies are proposed to shift this negative association, none has been successful due to the large influence of the environment (Monaghan et al., 2001; Oury et al., 2003; Iqbal et al., 2007). This includes the variation in cropping season (primarily the effect of different solar radiation and temperature), management, and cultural practices (plant density, rate and stime of nitrogen fertilization, water, and weed management). Studies have shown that elevated temperature progressively increased protein content in a japonica variety (Fujisaka 5), more than that of an Indica variety (IR20) which had a very low effect (Resurreccion et al, 1997).

The nitrogen assimilation and synthesis of amino acids requires ATP and NADP(H) and therefore competes with photosynthesis. It was shown that optimum light intensities stimulate the translation of nitrate reductase transcripts and as a result, high rates of nitrogen assimilation occur during the first part of the day like carbon assimilation (Scheible et al., 1997). Knowing metabolic cross paths in nitrogen and carbon assimilation will better understand the association between GPC and yield. Carbon and nitrogen reduction processes compete for electrons from the same source, that is the photochemical reaction in the chloroplast lamellar system. Consequently, depending on enzymatic efficiency or substrate availability, an increase in one metabolism could cause the other to decline. This association was stronger in high-yielding varieties due to the selection of genotypes only for yield. However, light is an abundant resource in tropical countries, whereas nitrogen is not. One-half of this light is photosynthetically active radiation and only a small fraction of this is utilized for net canopy photosynthesis resulting in very low radiation use efficiency (Slattery and Ort, 2021). Breeding programs determined to improve the use efficiency of these resources can help develop sustainable varieties that can break this trade-off and have higher protein content and yield. For this to happen it is crucial to understand the physiological basis of this trade-off between carbon and nitrogen assimilation which is very limited.

Plants that can uptake more nitrogen with help of transporters can have more nitrogen in the canopy and will be remobilized to the harvested organ as a useful protein (Parry and Hawkesford, 2010). In this regard, our hypothesis was that, constraints in nitrogen assimilation can be understood when carbohydrates are abundant. Hence, we screened the high-yielding rice germplasm lines for grain protein content. Two genotypes with similar assimilation but differing in grain protein content were selected for further experiments. Since reduced light intensity has been shown to decrease both carbon and nitrogen assimilation due to less availability of reductants, low light intensity was given to a set of plants and compared with ambient grown plants grown in ambient light intensity. This study aimed to understand how a genotype with better carbon and nitrogen assimilation manages the flow of reductants under low light conditions compared to a genotype with better carbon assimilation but lower nitrogen assimilation.

Plant material and growth conditions:

Ten rice genotypes were screened for grain protein content (GPC) and photosynthetic rate. Two contrasting rice germplasm lines GEN_RIC 812 (high grain protein content, GPC) and GEN_RIC 774 (low GPC) were used for the study (Supplementary Tables 1 and 2). Pre-germinated seeds were sown in the pots containing 25 Kg soil and farm yard manure (3:1). The seedlings were grown under normal ambient conditions (~ 1300 µmol m^− 2 s^− 1 of light intensity) for 21 days, later one set was covered with a shade net to get ~ 300 µmol m^− 2 s^− 1 of light intensity. Six plants were maintained for each treatment and for each genotype. Plants under low light condition were maintained until maturity. The recommended dose of fertilizers was applied similarly for both treatments. Nitrogen was applied in three spilt doses (at sowing, 21 days after sowing, and at the booting stage). Phosphorous and potassium were applied at the time of sowing.

Chlorophyll content:

Chlorophyll content of the leaf was measured using SPAD-502 (Soil Plant Analysis Development) chlorophyll meter. Readings were taken from the top, middle and bottom leaves in five replicates during bright sunshine hours. All the technical and biological replicates were pooled to arrive at SPAD value (Vijayaraghavareddy et al., 2020).

Gas exchange measurements:

Leaf photosynthesis of plants was measured during the booting stage from the mid-widest portion of fully opened flag leaves using LI-6400XT portable photosynthesis system (LI6400, LI-COR, Inc). Measurement of steady-state photosynthesis was done by giving a photosynthetic photon flux density (PPFD) of 1300 µmol m^− 2 s^− 1 for plants grown under ambient light and 300 µmol m^− 2 s^− 1 for low light-grown plants with 90% red light, 10% blue light, and 400 ppm CO₂ while maintaining a leaf temperature of 28°C.

Light response curves were plotted by measuring CO₂ assimilation rate at different light intensities of 50, 30, 50, 70, 100,150, 200, 500, 1000, 1500 and 2000 µmol m^− 2 s^− 1 and had a 120-second minimum wait time and a 200-second maximum wait time (Supplementary Fig. 1). Before altering the curves, it was made to match the CO₂ concentrations of the sample and reference samples to 400 ppm. A_sat stands for CO₂ assimilation rate at the maximum light intensity. Quantum efficiency (Ф_CO2) was computed from the initial slope of the light response curve (50 to 200 µmol m^− 2 s^− 1) (Vijayaraghavareddy et al., 2022).

CO₂ response curves were plotted by measuring CO₂ assimilation rate at different CO₂ concentrations of 50, 100, 150, 200, 250, 400, 700, 800, 900, and 1000 ppm (Supplementary Fig. 2). The illumination of light was fixed to 1300 µmol m^− 2 s^− 1 for ambient light treatment and 300 µmol m^− 2 s^− 1 for low light treatment. Maximum assimilation achieved at the highest CO₂ level was calculated (A_max). The carboxylation efficiency (CE) of the genotype was determined by the initial slope of the carbon dioxide response curve (50 ppm-200 ppm).

The insensitivity of photosynthesis to CO₂ or its decline with increasing CO₂ has been connected to a restriction on the export of carbon from the Calvin-Benson cycle and it is termed the triose phosphate utilization (TPU) limit. Formula used to (The Farquhar, von Caemmerer, and Berry model of photosynthesis, 1980) to determine the TPU limit was;

$$\mathbf{T}\mathbf{P}\mathbf{U}=\frac{A\text{g}+Rd}{3}$$

Where A_g= Gross assimilation rate,

R _d= Day respiration.

Chlorophyll fluorescence:

A pulse amplitude modulation chlorophyll fluorometer was used to detect the chlorophyll fluorescence in dark-adapted leaves for 30 minutes (LICOR 6400XT). The basal fluorescence, Fo was measured in dark-acclimated leaves by the input of weak modulated light of < 0.15 µmol m^− 2 s^− 1, and the maximum fluorescence, Fm was measured after providing a saturating pulse of > 8000 µmol m^− 2 s^− 1 light. The parameters for photochemical (qP) and non-photochemical quenching (NPQ) were determined.

Nitrate reductase activity:

Nitrate reductase activity was measured using a modified version of Hageman and Hucklesby's method (1971). Rice root tissue was homogenized with ice-cold 25mM Potassium phosphate buffer (10mM KNO₃ and 0.5mM EDTA- pH 7.3 at 30℃). 20 µl of extracts were added to 380 µl of a reaction mixture containing 50mM KH₂PO₄, 20mM KNO_3, and 0.35mM NADH, and then the mixture was incubated at 30°C for 30 min. This process was stopped by adding 300µl of 0.02% (w/v) N-1-naphthyl ethylenediamine dihydrochloride and 300µl of 1% (w/v) sulfanilamide in 3M HCl. The absorbance was measured at 540nm, and NaNO₂ was used to plot the standard curve.

Nitrite reductase activity:

Nitrite reductase activity was evaluated in accordance with Joy and Hageman (1966). Rice leaf tissue was homogenized with ice-cold 50mM Tris-Hcl₂ (pH 7.5) with adding 40mM Phenyl methyl sulfonyl fluoride (PMSF) and 2% (w/v) polyvinyl pyrrolidone (PVP). 25µl of this extract was added after 150 µl of 57.4 mM Na₂S₂O₄ in 290 mM NaHCO₃ was added to a 500 µl reaction buffer (50 mM KH₂PO₄, pH 7.5, 1 mM NaNO₂, 1 mM methyl viologen). After incubating the mixture at 30°C for 60 minutes, 40µl of this mixture is transferred to a fresh tube and mixed with 360µl of water 300µl of 0.02% (w/v) N-1-naphthyl ethylenediamine dihydrochloride and 300µl of 1% (w/v) sulfanilamide in 3M HCl were added to this tube and pink colour develops. The reaction mixture's absorbance at 540 nm was measured, and NaNO₂ was used to plot the standard curve.

Gene expression analysis:

Total RNA was extracted from the roots and leaves of different treatments using the phenol-chloroform method (Vijayaraghavareddy et al., 2020) to assess the expression levels of nitrogen transport-related genes involved in nitrogen absorption, translocation, and remobilization mechanisms. Root samples were collected at flowering stage to check the expression of NRT1.0, NRT2.1 and NRT2.3. While leaf samples were collected at same stage to analyze the expression of NPF2.4, NPF2.2 and NRT1.1b. The quantity and purity of RNA were verified at the A_260/280 and A_260/230 wavelength ratios using the NanoDrop® ND- 1000 spectrophotometer. The cDNA was generated from total RNA by reverse transcription using oligo (dT) primers and M-MLV reverse transcriptase. A quantitative RT-PCR reaction was carried out using the Takara SYBR Green qPCR Kit and the iQ5 real-time PCR detection apparatus (Bio-Rad). The entire reaction volume was 10 µl. The following cycling conditions were used for the PCR programme: 94°C for three minutes, followed by 25 cycle programmes (94°C for 30 seconds, followed by 30s at 52 to 58°C, and 5 minutes at 72°C per cycle). Ubiquitin was used as an endogenous reference gene to normalize the results. The relative expression of the nitrogen transporter genes was calculated using the 2^−∆∆Ct method (Livak and Schmittgen, 2001). Supplementary Table 3, provides information on the primers used for genes involved in nitrogen transport that were validated using quantitative RT-PCR.

Measurement of morpho-physiological parameters:

During the end of the vegetative stages of plant growth and development, measurements of the plant height, tiller count, leaf width, specific leaf area, chlorophyll content, and days to 50% flowering were made. Total leaf area was measured as the product of specific leaf area (SLA) and total leaf weight. When plant was harvested, the above-ground biomass was separated into stems, leaves, leaf sheaths, and roots. All the plant parts were oven dried at 65 ºC until they attain constant weight and weighed separately. A total number of filled seeds was weighed to get the yield per pot. One hundred filled seeds were separated to obtain 100-seed weight.

Total grain protein content:

Total grain protein content was measured by following the Da-Gin Lin and Chang-Sheng Wang (2014) method of extraction of total protein fractions from the rice grains. The crude protein was extracted from 0.2g grains using 2ml extraction buffer containing, tris base, 40mM PMSF. The homogenate is centrifuged for 20 min at 13,500 rpm at 4℃ and the supernatant was incubated in precipitation buffer overnight. The next day the precipitated proteins were again centrifuged for 20 min at 13,500 rpm at 4℃, and the pellet formed is washed with precipitation buffer twice and once with ice-cold 100% acetone containing 0.7 µl 2-mercaptoethanol by centrifuging at 8000 rpm for 5min at 4℃ and the pellets were dried in the Speed vac system for about 2–3 minutes and resuspended in lysis buffer and the proteins are quantified from the absorption at 540 nm using Bradford’s reagent (Bradford, 1976).

Differences in chlorophyll content and photosynthetic parameters between low and high protein genotypes.

Competition for electrons between carbon and nitrogen assimilation processes is expected to bring in the trade-off between grain protein content (GPC) and grain yield. Two rice genotypes with contrasting grain protein content and similar yield and assimilation were utilized in this study (Supplementary Tables 1 and 2). To examine the relevance of chloroplast electron transport chain and hence the role of electron source on carbon and nitrogen assimilations, the selected genotypes were grown in low light and compared with that of ambient light intensities (~ 1300 µmol m^− 2 s^− 1 of light intensity).

The total chlorophyll content of GEN_RIC 774 was significantly lower than that of GEN_RIC 812 under ambient light. In both the genotypes, an increase in chlorophyll content was found under low light. However, in GEN_RIC 812, this increase was not statistically significant. Carbon assimilation rate at maximum CO₂ (A_max) was significantly higher in GEN_RIC 774 (Fig. 1b). Corroborating with the A_max data, the carboxylation efficiency (CE) determined from the initial slope of CO₂ response curve was higher in GEN_RIC 774 (Fig. 1c). However, a distinctly different trend was observed for CE in low light among these genotypes. CE of GEN_RIC 812, the high GPC type was lower under low light while it significantly increased in GEN_RIC 774. These data indicates that the low GPC line had higher carbon assimilation capacity. The quantum efficiency (ɸ_CO2), computed from the initial slope of light response curves was higher in GEN_RIC 812 which was comparable under ambient and low light condition. The reduction in ɸ_CO2 under low light was significant for genotype GEN-RIC 774 (Fig. 1d). Similar trend was observed for A_sat (Fig. 1e). The interesting phenomenon was the differences in triose phosphate utilization (TPU), the high GPC line, GEN_RIC 812 recorded a significantly lower TPU under both light treatments compared to GEN-RIC 774 (Fig. 1f). However, no difference between treatments was observed for TPU in both the genotypes.

In order to assess the impact of electron source, chlorophyll fluorescence measurements were conducted. The findings presented in Fig. 2 indicated that Fv/Fm, qP, and ɸPSII were elevated under low light intensity, while NPQ decreased under such condition. Notably, there were no differences observed in qP between the genotypes under ambient conditions. However, the GEN_RIC 774 genotype exhibited higher values of Fv/Fm and NPQ, while the GEN_RIC 812 genotype displayed marginally higher ΦPSII.

Expression of genes involved in nitrogen metabolism.

We examined the expression of various nitrogen transporters in root tissue (NRT1.0, NRT2.1 and NRT2.3) and in leaf tissue (NPF2.4, NPF2.2 and NRT1.1b) (Fig. 3). The NRT1.0 dual affinity transporter, localized in the root, exhibited significant induction under low light conditions for both genotypes. No differences were observed between the genotypes under ambient conditions. In contrast, the expression of the high-affinity transporter NRT2.1 showed distinct patterns between the genotypes. Low light conditions resulted in lower expression of NRT2.1 in the GEN_RIC 812, while higher expression was observed in the GEN_RIC 774 compared to the ambient condition. Another high-affinity transporter, NRT2.3, displayed higher expression in the GEN_RIC 812 genotype compared to the GEN_RIC 774. Moreover, significant difference for expression of NRT2.3 between the treatments was only found in GEN_RIC 812. However, under ambient conditions, the expression of both NRT2.1 and NRT2.3 were significantly higher in GEN_RIC 812 than GEN_RIC 774. The low- affinity transporter NPF 2.4 was shown higher expression in GEN_RIC 812 under ambient condition contradicting its higher expression in GEN_RIC 774 under low light. Whereas, another low-affinity transporter NPF2.2 showed no significant differences between genotypes and treatments. Additionally, we analysed the expression of NRT1.1b, involved in the remobilization of stored nitrogen from the shoot to reproductive tissue (Fig. 2). The expression pattern of NRT1.1b was similar to that of NRT2.3, but unlike NRT2.3, no differences were found between the treatments for both the genotypes. After thoroughly analysing the differences in the relative expression of genes between the genotypes and treatments, we further checked the enzyme activities that are involved in nitrogen assimilation.

Nitrate and nitrite reductase activity under ambient and low light condition:

Nitrate reductase and nitrite reductase are two crucial enzymes involved in nitrogen assimilation. The study found no variation in nitrate reductase activity among the genotypes and treatments when measured in root tissues (Table 1). However, under ambient conditions, the GEN_RIC 812 genotype exhibited higher nitrite reductase activity in leaves compared to the GEN_RIC 774 genotype. Interestingly, no significant differences in nitrite reductase activity between the genotypes were observed under low light treatment (Table 1).

Table 1

**Nitrate reductase and Nitrite reductase enzyme activity** (**µmol NO**₂^-g^-1FW). Different letters indicate significance (P < 0.05) from the LSD post-hoc test determined using two-way ANOVA with genotypes × treatments interactions. Note: Nitrite reductase values are the representation of leftover NO₂^- in the samples after enzymatic conversion of NO₂^- to NH₄⁺, therefore lesser the values, the more is the activity of enzyme.
Genotypes	Nitrate reductase enzyme activity (µmol NO₂⁻g⁻¹ FW)		Nitrite reductase enzyme activity (µmol NO₂⁻g⁻¹ FW)
	Low Light	Ambient	Low Light	Ambient
GEN_RIC 812	1.280 ± 0.052	1.337 ± 0.049	3.463 ± 0.051 ^ab	3.340 ± 0.062 ^b
GEN_RIC 774	1.333 ± 0.021	1.340 ± 0.020	3.480 ± 0.030 ^ab	3.610 ± 1.32 ^a

Variability in morpho-physiological traits under different light conditions

Various morpho-physiological parameters were measured, including leaf width, total leaf area, tiller number, biomass, days to fifty percent flowering, hundred seed weight, and grain protein content (Fig. 4), all of which contribute to total grain yield. The leaf width of plants grown under low light did not differ significantly from that of ambient light grown plants in both genotypes (Fig. 4a). However, the leaf width of GEN_RIC 812 was notably higher than that of GEN_RIC 774 in ambient conditions. The leaf area of low light-grown plants in GEN_RIC 774 was significantly lower compared to that of ambient light grown plants, but no difference in tiller number was observed (Fig. 4c). Tiller number also showed no significant difference in GEN-RIC 812 between treatments. While there was no difference in biomass between treatments for GEN_RIC 812, indicating consistent canopy cover. A significant reduction in biomass was observed under low light conditions for GEN_RIC 774 (Fig. 4d). The time taken for plants to reach 50% flowering was longer for low light-grown plants of GEN_RIC 812 compared to its ambient light (Fig. 4e), while no difference in flowering time was observed for GEN_RIC 774.

The high protein genotype, due to its higher biomass in the ambient condition, was capable of filling the grains, resulting in a yield (Fig. 5a). However, in GEN_RIC 774, no grain filling was observed. Interestingly, the 100-grain weight increased under low light conditions in GEN_RIC 812 (Fig. 5b). Moreover, in ambient condition GEN_RIC 812 had a higher seed weight compared to GEN_RIC 774. There were no significant differences in grain protein content (GPC) between treatments within GEN_RIC 812 (Fig. 5c). Consistent with our previous findings (Supplementary table 1), GEN_RIC 812 exhibited higher GPC compared to GEN_RIC 774 genotype. Overall, under ambient light conditions, GEN_RIC 812 exhibited lower CE, TPU, Ф_CO2 and higher NiR activity with increased expression of nitrate transporters (Fig. 6). Whereas, GEN_RIC 774 showed higher NPQ, CE, A_max, and TPU, depicting increased carbon assimilation than nitrogen assimilation.

Carbon and nitrogen are the most important elements, also the limiting factors for plant growth, crop yield, and grain protein content (Lawlor et al., 2001). Assimilation of carbon and nitrogen in plants involves complex processes and is affected by many genetic and epigenetic factors. To examine the effect of these factors in contrasting genotypes, chloroplast electron transport rate (different intensities of light), gas exchange and nitrogen metabolism were studied in this experiment.

Effect of light on chlorophyll content in contrast genotypes:

Earlier studies have shown that decreasing light levels resulted in increased chlorophyll and carotenoid concentrations in sage (Rezai et al., 2018). This is to increase the plant’s ability to efficiently use the captured light and to maximize the capacity of the light-harvesting complex with decreased maintenance cost under low light (Lei et al., 1996; Dai et al., 2009). This was evident in our study, where both the genotypes had higher chlorophyll content under low light treatment as indicated by SPAD values (Fig. 1a). Excess light causes greater degradation of chloroplast, leading to decreased levels of chlorophyll (Goncalves et al., 2005). In our experiment, GEN_RIC 812 had higher chlorophyll content under ambient light condition as it was avoiding degradation of chlorophyll by utilizing excess light. This is also evident by higher ΦPSII and lower NPQ (Fig. 2c,d).

Varying light intensity differentially affects photosynthesis in contrasting genotypes.

It is well known that the second largest sink for photosynthetic energy is nitrogen assimilation in higher plants. Both the carbon and nitrogen assimilation use light energy and it was estimated that around 10% of electron flux in photosynthesizing leaves is used in NO₃⁻ reduction (Foyer et al., 2001). Hence, looking for a genotype with better photosynthesis and nitrogen assimilation through better radiation use efficiency is important. In this study, low grain protein genotype, GEN_RIC 774 showed higher CE as well as A_max in both low light and ambient light intensity (Fig. 1b and Fig. 1c), indicating the dominance of photosynthesis over nitrogen metabolism. This also could be due to the lower quantum efficiency of GEN_RIC 774 leading to increased competition between photosynthesis and nitrogen assimilation. Φ_CO2 of GEN_RIC 812 was higher under both light treatments (Fig. 1e), implying it is efficiently using all photons incident on it, however, there is no increase in photosynthesis, which is evident from low CE and A_max, indicating there is allocation of electrons to alternative sinks other than CO₂ reduction. The other possibility for GEN_RIC 812 to have high protein as well as high photosynthesis could be due to higher TPU limitation (Fig. 1d). When the TPU is limited, indicating lower photosynthesis (McClain and Sharkey, 2019), hence the allocation might have diverted to nitrogen assimilation in GEN_RIC 812. However, in GEN_RIC 774 under ambient conditions, electrons are quenched in the form of heat as indicated by higher NPQ rather than utilizing it for other processes (Fig. 2c). These results indicate that, though GEN_RIC 774 had the higher photosynthesis, it lacks the utilization of light energy. Higher chlorophyll content in GEN_RIC 812 leads to the presence of excess irradiated energy, which cannot be used for CO₂ assimilation alone. This hypothesis is supported by higher nitrite reductase activity in GEN_RIC 812 than in GEN_RIC 774 (Fig. 6). A_sat of GEN_RIC 812 under low light was higher compared to GEN_RIC 774 (Fig. 1f), indicating that high grain protein genotypes grown under low light were able to photosynthesize better even when there was a sudden flux of high light intensity (2000 µmol m^− 2 s^− 1).

Contribution of nitrogen transporters to high protein content.

Various transporters are involved in the uptake of nitrate and ammonia from soil and mobilize it to different parts of the plant. These molecules are used for the synthesis of amino acids in later stages. The abundance of transporters is positively correlated with protein content. We estimated the mRNA expression of these transporters in different plant parts to understand the complexity of nitrogen movement throughout the plant (Fig. 3). NRT2.3 is a plasma membrane-localized nitrate transporter expressed in parenchyma cells around the root xylem and expressed in shoot phloem which is responsible for long-distance NO₃⁻ transport (Tang et al., 2012; Fan et al., 2016a). This transporter’s expression was higher in roots of GEN_RIC 812 indicating the higher nitrate transport from the root to upper parts of plant. A spliced form of NRT2.3 i.e., NRT2.3a present in plasma membrane of shoot tissues helps to uptake and translocation of nitrate in the shoot tissues (Fan et al., 2016b). Enhanced accumulation of this gene was correlated with improved yield and biomass in tomato (Fu et al., 2015). It was expressed more in GEN_RIC 774 under ambient light to contribute nitrogen to biomass accumulation than to plant proteins. NRT2.1 expression is influenced by glucose content and nitrate availability in the plant roots (De Jong et al., 2013). Higher expression of NRT2.1 in the high protein type is by the virtue of higher nitrate absorbed from the soil (Fig. 6). NPF2.4 is involved in source to sink redistribution and long-distance transport of NO₃⁻ to various parts of plants (Xia et al., 2015). Higher expression of NPF2.4 in GEN_RIC 812 under optimal light condition ensures efficient transport and redistribution of nitrate to sink organs. Since high NiR activity aids in the synthesis and accumulation of amino acids and peptides in leaves, the expression of NRT1.1b which is involved in the remobilization of N into grains was also found to be higher in GEN_RIC 812. In a nutshell, by adopting various strategies like increased uptake, translocation, remobilization, and increased NiR activity GEN_RIC 812 genotype had higher grain protein content (Fig. 6).

Acknowledgment:

This work was supported by a project entitled “Strategic understanding of mechanisms that restrict grain yield, grain protein and quality in rice through an interface between clinical nutrition and agriculture research” funded by Department of Biotechnology (BT/PR42156/AGIII/103/1352/2021), Government of India. We thank IRRI, Philippines 3K-Panel, for providing rice seeds.

Author Contributions:

SS conceived and supervised the project; SS and PV designed the research; NS and RM performed the experiments and data analysis, and wrote the draft; PV, RA and PC revised the manuscript.

Funding:

The studies by the authors were partly supported by the project “Strategic understanding of mechanisms that restrict grain yield, grain protein and quality in rice through an interface between clinical nutrition and agriculture research” funded by Department of Biotechnology (BT/PR42156/AGIII/103/1352/2021), Government of India.

Author information

Authors and Affiliations:

Department of Crop Physiology, University of Agricultural Sciences, GKVK, Bengaluru, India.

Nagaraju Spoorthi, Ramesh Mohan, Preethi Vijayaraghavreddy, Ramachandra Abhishree, Purushothama Chaitanya, and Sheshshayee Sreeman.

Corresponding author

Correspondence to Sheshshayee Sreeman, [email protected].

Competing interests:

The authors have no relevant financial or non-financial interests to disclose.

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Plausible Photosynthetic Parameters that Contribute to Differences in Grain Protein Content among Rice Genotypes

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Abstract

Aim

Methods

Results

Conclusions

Figures

Introduction

Materials and methods

Plant material and growth conditions:

Chlorophyll content:

Gas exchange measurements:

Chlorophyll fluorescence:

Nitrate reductase activity:

Nitrite reductase activity:

Gene expression analysis:

Measurement of morpho-physiological parameters:

Total grain protein content:

Results

Nitrate and nitrite reductase activity under ambient and low light condition:

Variability in morpho-physiological traits under different light conditions

Discussion

Effect of light on chlorophyll content in contrast genotypes:

Declarations

References

Supplementary Files

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