Modern rice production faces the dual challenges of increasing grain yields while reducing inputs of chemical fertilizer.  However, the disequilibrium between the nitrogen (N) supplement from the soil and the demand for N of plants is a serious obstacle to achieving these goals.  Plant-based diagnosis can help farmers make better choices regarding the timing and amount of topdressing N fertilizer.  Our objective was to evaluate a non-destructive assessment of rice N demands based on the relative SPAD value (RSPAD) due to leaf positional differences.  In this study, two field experiments were conducted, including a field experiment of different N rates (Exp. I) and an experiment to evaluate the new strategy of nitrogen-split application based on RSPAD (Exp. II).  The results showed that higher N inputs significantly increased grain yield in modern high yielding super rice, but at the expense of lower nitrogen use efficiency (NUE).  The N nutrition index (NNI) can adequately differentiate situations of excessive, optimal, and insufficient N nutrition in rice, and the optimal N rate for modern high yielding rice is higher than conventional cultivars.  The RSPAD is calculated as the SPAD value of the top fully expanded leaf vs. the value of the third leaf, which takes into account the non-uniform N distribution within a canopy.  The RSPAD can be used as an indicator for higher yield and NUE, and guide better management of N fertilizer application.  Furthermore, we developed a new strategy of nitrogen-split application based on RSPAD, in which the N rate was reduced by 18.7%, yield was increased by 1.7%, and the agronomic N use efficiency was increased by 27.8%, when compared with standard farmers’ practices.  This strategy of N fertilization shows great potential for ensuring high yielding and improving NUE at lower N inputs.

Integrative cultivation practices (ICPs) are essential for enhancing cereal yield and resource use efficiency.  However, the effects of ICP on the rhizosphere environment and roots of paddy rice are still poorly understood.  In this study, four rice varieties were produced in the field.  Each variety was treated with six different cultivation techniques, including zero nitrogen application (0 N), local farmers’ practice (LFP), nitrogen reduction (NR), and three progressive ICP techniques comprised of enhanced fertilizer N practice and increased plant density (ICP1), a treatment similar to ICP1 but with alternate wetting and moderate drying instead of continuous flooding (ICP2), and the same practices as ICP2 with the application of organic fertilizer (ICP3).  The ICPs had greater grain production and nitrogen use efficiency than the other three methods.  Root length, dry weight, root diameter, activity of root oxidation, root bleeding rate, zeatin and zeatin riboside compositions, and total organic acids in root exudates were elevated with the introduction of the successive cultivation practices.  ICPs enhanced nitrate nitrogen, the activities of urease and invertase, and the diversity of microbes (bacteria) in rhizosphere and non-rhizosphere soil, while reducing the ammonium nitrogen content.  The nutrient contents (ammonium nitrogen, total nitrogen, total potassium, total phosphorus, nitrate, and available phosphorus) and urease activity in rhizosphere soil were reduced in all treatments in comparison with the non-rhizosphere soil, but the invertase activity and bacterial diversity were greater.  The main root morphology and physiology, and the ammonium nitrogen contents in rhizosphere soil at the primary stages were closely correlated with grain yield and internal nitrogen use efficiency.  These findings suggest that the coordinated enhancement of the root system and the environment of the rhizosphere under integrative cultivation approaches may lead to higher rice production.

Recently developed ‘super’ rice cultivars with greater yield potentials often suffer from the problem of poor grain filling, especially in inferior spikelets.  Here, we studied the activities of enzymes related to starch metabolism in rice stems and grains, and the microstructures related to carbohydrate accumulation and transportation to investigate the effects of different water regimes on grain filling.  Two ‘super’ rice cultivars were grown under two irrigation regimes of well-watered (WW) and alternate wetting and moderate soil drying (AWMD).  Compared with the WW treatment, the activities of ADP glucose pyrophosphorylase (AGPase), starch synthase (StSase) and starch branching enzyme (SBE), and the accumulation of non-structural carbohydrates (NSCs) in the stems before heading were significantly improved, and more starch granules were stored in the stems in the AWMD treatment.  After heading, the activities of α-amylase, β-amylase, sucrose phosphate synthase (SPS) and sucrose synthase in the synthetic direction (SSs) were increased in the stems to promote the remobilization of NSCs for grain filling under AWMD.  During grain filling, the enzymatic activities of sucrose synthase in the cleavage direction (SSc), AGPase, StSase and SBE in the inferior spikelets were increased, which promoted grain filling, especially for the inferior spikelets under AWMD.  However, there were no significant differences in vascular microstructures.  The grain yield and grain weight could be improved by 13.1 and 7.5%, respectively, by optimizing of the irrigation regime.  We concluded that the low activities of key enzymes in carbon metabolism is the key limitation for the poor grain filling, as opposed to the vascular microstructures, and AWMD can increase the amount of NSC accumulation in the stems before heading, improve the utilization rate of NSCs after heading, and increase the grain filling, especially in the inferior spikelets, by altering the activities of key enzymes in carbon metabolism.

Aligning leaf nitrogen (N) distribution to match the light gradient is crucial for maximizing canopy dry matter production (DMP) and improving N utilization efficiency.  However, the relationship between the gradient of root-derived cytokinins and N distribution in rice leaves, along with its impact on DMP and the underlying mechanisms, remains poorly understood.  A two-year field experiment was conducted using two japonica N-efficient varieties (NEVs) and two japonica N-inefficient varieties (NIVs) under four different N rates (0, 90, 180 and 360 kg N ha⁻¹). These selected varieties exhibited similar values in the coefficient of light extinction (K_L).  Results showed that, at lower N rates (0-180 kg N ha⁻¹), the NEVs exhibited greater dry matter weight at maturity, higher grain yield and improved internal N use efficiency (IE_N), compared to the NIVs, despite possessing comparable total N uptake.  Compared with the NIVs, the NEVs exhibited a more pronounced nitrogen distribution gradient in leaves, as indicated by the coefficient of nitrogen extinction (K_N) values during the middle and early grain filling stages.  This enhanced gradient led to improved coordination between light and nitrogen, resulting in greater photosynthetic production, particularly at lower N rates. Furthermore, the NEVs demonstrated a larger gradient of zeatin (Z)+zeatin riboside (ZR) in leaves (i.e., higher ratios of Z+ZR levels between upper and lower leaves), enhanced expression levels of genes related to N export in lower leaves and Z+ZR loading in root, respectively, elevated enzymes activities related to N assimilation in upper leaves, in relative to the NIVs.  Correlation and random forest analyses demonstrated a strong positive correlation between Z+ZR gradient, K_N, and DMP, and the gradient facilitated the export of N from lower leaves and its assimilation in upper leaves, contributing significantly to both K_N and DMP.  This process was closely linked to root activity, including root oxidation activity, root Z+ZR content, and Z+ZR loading capacity, as confirmed by applying an inhibitor or a promoter of cytokinins biosynthesis to roots.  Interestingly, at the N rate of 360 kg N ha⁻¹, both NEVs and NIVs showed indistinguishable plant traits, achieving a super high-yielding level (over 10.5 t ha⁻¹) but with remarkably low IE_N.  The results suggest that increasing Z+ZR gradient can improve K_N and DMP, where it needs to maintain higher root activity, thus leading to high yield and high IE_N.  Further research is needed to explore and develop cultivation practices with reduced N to unlock the super high-yielding potential of the NEVs.

Alternate wetting and soil drying irrigation (AWD) technique plays crucial influences on grain quality in rice (Oryza sativa L.).  Lipids are the third abundant constitutes besides starch and proteins in rice grains and closely related with grain quality.  However, it remains unclear about the changes in lipids profiling under different AWD regimes.  This study set up three irrigation regimes including conventional irrigation (CI), alternate wetting and moderate soil drying irrigation (AWMD) and alternate wetting and severe soil drying irrigation (AWSD), and employed the untargeted lipidomics approach to explore lipidome changes in milled rice of Yangdao 6 (YD6), and rice cooking and eating quality was also analyzed.  The results showed that 7 lipid classes, 55 lipid subclasses, and a total of 1,086 lipid molecular species were identified.  Compared with CI regime, AWMD regime mainly altered these lipid subclasses consisting of TG, Cer, DG, BisMePA, PC, PE, MGDG, and DGDG in milled rice and improved cooking and eating quality of rice, while AWSD regime distinctly changed these lipid subclasses like TG, Cer, DG, PC, PE, Hex1Cer, DGDG, and BisMePA, and degraded cooking and eating quality of rice.  Specifically, AWMD regime most significantly altered the expressions of these lipid molecules including DGDG(18:0_18:2), DGDG(16:0_14:0), PC(33:1), Cer(t17:0_26:0), and Cer(t17:0_16:0), while AWSD regime most obviously influenced expressions of TG(6:0_14:0_18:3), PC(41:1), TG(19:1_18:4_18:4), Hex1Cer(d18:2_24:0+O), and Hex1Cer(d18:2_24:1).  These 10 altered lipid molecules in milled rice can be preferentially used to focus on their relationships with grain quality in rice.

Alternate wetting and drying irrigation (AWD) plays crucial roles in regulating the cooking and eating quality of rice (Oryza sativa L.).  However, it remains unclear about how AWD influences rice cooking and eating quality.  Lipid and free fatty acids in grains are positively related with cooking and eating quality of rice. In this study, Yangdao 6 (YD6, a conventional taste indica inbred) and Nanjing 9108 (NJ9108, a superior taste japonica inbred) were planted in the field with conventional irrigation (CI), alternate wetting and moderate drying irrigation (AWMD), and alternate wetting and severe drying irrigation (AWSD) from 10 days after transplanting to maturity.  The relationships between the biosynthesis of lipid and free fatty acids in grains and cooking and eating quality of rice were investigated.  Compared with CI treatment, the AWMD significantly increased contents of lipid, total free fatty acids (TFFAs), free unsaturated fatty acids (FUFAs), linoleic acid, and oleic acid in milled rice by promoting activities of the enzymes associated with lipid synthesis, while AWSD regime exerted the opposite effect.  Correlation analysis showed that higher contents of lipid, TFFAs, FUFAs, linoleic acid, and oleic acid were more beneficial to improvement in rice cooking and eating quality.  These results demonstrated that AWMD regime could improve cooking and eating quality of milled rice by optimizing lipid and fatty acid synthesis in rice grains.

This study examined the involvement of cytokinins in the process by which moderate water limitation (MWL) mediates nitrogen (N) remobilization from source to sink during the grain-filling phase in wheat.  Field experiments were performed using N application rates of low (LN), medium (MN), and high (HN).  Two soil moisture regimes were implemented for each N rate: conventional well-watered (CWW) and MWL post anthesis. The MWL application optimized N, total free amino acids (FAA), trans-zeatin (Z)+trans-zeatin riboside (ZR) reallocation from the source organs (stems and leaves) to the sink organ (spikes) in wheat.  Compared to those in the CWW regime, the activities of proteolytic enzymes, including endopeptidase, carboxypeptidase and aminopeptidase within stems and leaves, and the expression levels of total FAA transporter genes in spikes were significantly elevated in the MWL regime, showing a close correlation with the Z+ZR levels in the spikes.  Application of kinetin to stems and leaves significantly inhibited proteolytic enzyme activity, promoting N retention in stems and leaves, decreasing N accumulation in the sink organ, and reducing the N harvest index.  In contrast, the applying kinetin to spikes significantly upregulated expression levels of FAA transporter genes, reducing N retention in stems and leaves, increasing N accumulation in the sink organ, and raising the N harvest index.  Such facilitation induced by the MWL in remobilization of N from source to sink was greater at HN than at LN or MN.  Results demonstrate that post-anthesis MWL can significantly intensify the remobilization of N from source to sink, while also synergistically enhancing grain yield and N use efficiency through strategically redistributing cytokinins (Z+ZR) between source and sink in wheat.

Globally recurrent extreme high temperature (HT) events severely limit rice production.  This study investigated whether a controlled moderate soil drying (MD) could replace the conventional well-watered (WW) regime to more effectively mitigate HT stress on pistil fertilization in photo-thermosensitive genetic male-sterile (PTGMS) rice, and examined the role of brassinosteroids (BRs).  Two PTGMS rice varieties were cultivated under normal temperature (NT) and HT conditions, paired WW and MD strategies during anthesis.  In conventional WW regime, waterlogging reduces BRs levels in roots and pistils due to excessive decomposition, weakening active water uptake driven by root activity and failing to alleviate transpiration-pulled passive water extraction hampered by restricted stomatal openings.  Thereby, it causes water imbalance in plants and weakened pistil function due to a suppressed ascorbate-glutathione (AsA-GSH) cycle and hyperactive nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity.  This exacerbates pistil fertilization impairment and hybrid seed yield loss under HT stress.  Conversely, by promoting BR synthesis and inhibiting its decomposition in roots and pistils, the MD strategy enhanced root activity and transpiration-driven water uptake.  It maintained plant water balance and supported pistil function through suppressed NOX activity and an enhanced AsA-GSH cycle-driven redox homeostasis.  Thus, it mitigated HT-induced pistil fertilization impairment and hybrid seed yield loss.  The precise function of BRs in moderating the protective effects of MD against the detrimental impacts of HT stress on pistil fertilization in PTGMS rice was confirmed through genetic and chemical approaches.  Consequently, a controlled MD method proved to be more effective than the conventional WW regime in alleviating HT stress on pistil fertilization in PTGMS rice by promoting BR enhancement.