Agronomic nitrogen‐use efficiency of rice can be increased by driving OsNRT2.1 expression with the OsNAR2.1 promoter

The importance of the nitrate () transporter for yield and nitrogen‐use efficiency (NUE) in rice was previously demonstrated using map‐based cloning. In this study, we enhanced the expression of the OsNRT2.1 gene, which encodes a high‐affinity transporter, using a ubiquitin (Ubi) promoter and the ‐inducible promoter of the OsNAR2.1 gene to drive OsNRT2.1 expression in transgenic rice plants. Transgenic lines expressing pUbi:OsNRT2.1 or pOsNAR2.1:OsNRT2.1 constructs exhibited the increased total biomass including yields of approximately 21% and 38% compared with wild‐type (WT) plants. The agricultural NUE (ANUE) of the pUbi:OsNRT2.1 lines decreased to 83% of that of the WT plants, while the ANUE of the pOsNAR2.1:OsNRT2.1 lines increased to 128% of that of the WT plants. The dry matter transfer into grain decreased by 68% in the pUbi:OsNRT2.1 lines and increased by 46% in the pOsNAR2.1:OsNRT2.1 lines relative to the WT. The expression of OsNRT2.1 in shoot and grain showed that Ubi enhanced OsNRT2.1 expression by 7.5‐fold averagely and OsNAR2.1 promoters increased by about 80% higher than the WT. Interestingly, we found that the OsNAR2.1 was expressed higher in all the organs of pUbi:OsNRT2.1 lines; however, for pOsNAR2.1:OsNRT2.1 lines, OsNAR2.1 expression was only increased in root, leaf sheaths and internodes. We show that increased expression of OsNRT2.1, especially driven by OsNAR2.1 promoter, can improve the yield and NUE in rice.     

Concurrent activation of OsAMT1;2 and OsGOGAT1 in rice leads to enhanced nitrogen use efficiency under nitrogen limitation

Nitrogen (N) is a major factor for plant development and productivity. However, the application of nitrogenous fertilizers generates environmental and economic problems. To cope with the increasing global food demand, the development of rice varieties with high nitrogen use efficiency (NUE) is indispensable for reducing environmental issues and achieving sustainable agriculture. Here, we report that the concomitant activation of the rice (Oryza sativa) Ammonium transporter 1;2 (OsAMT1;2) and Glutamate synthetase 1 (OsGOGAT1) genes leads to increased tolerance to nitrogen limitation and to better ammonium uptake and N remobilization at the whole plant level. We show that the double activation of OsAMT1;2 and OsGOGAT1 increases plant performance in agriculture, providing better N grain filling without yield penalty under paddy field conditions, as well as better grain yield and N content when plants are grown under N llimitations in field conditions. Combining OsAMT1;2 and OsGOGAT1 activation provides a good breeding strategy for improving plant growth, nitrogen use efficiency and grain productivity, especially under nitrogen limitation, through the enhancement of both nitrogen uptake and assimilation.     

PAY1 improves plant architecture and enhances grain yield in rice

Plant architecture, a complex of the important agronomic traits that determine grain yield, is a primary target of artificial selection of rice domestication and improvement. Some important genes affecting plant architecture and grain yield have been isolated and characterized in recent decades; however, their underlying mechanism remains to be elucidated. Here, we report genetic identification and functional analysis of the PLANT ARCHITECTURE AND YIELD 1 (PAY1) gene in rice, which affects plant architecture and grain yield in rice. Transgenic plants over‐expressing PAY1 had twice the number of grains per panicle and consequently produced nearly 38% more grain yield per plant than control plants. Mechanistically, PAY1 could improve plant architecture via affecting polar auxin transport activity and altering endogenous indole‐3‐acetic acid distribution. Furthermore, introgression of PAY1 into elite rice cultivars, using marker‐assisted background selection, dramatically increased grain yield compared with the recipient parents. Overall, these results demonstrated that PAY1 could be a new beneficial genetic resource for shaping ideal plant architecture and breeding high‐yielding rice varieties.     

Improving rice tolerance to potassium deficiency by enhancing OsHAK16p:WOX11‐controlled root development

Potassium (K) deficiency in plants confines root growth and decreases root‐to‐shoot ratio, thus limiting root K acquisition in culture medium. A WUSCHEL‐related homeobox (WOX) gene, WOX11, has been reported as an integrator of auxin and cytokinin signalling that regulates root cell proliferation. Here, we report that ectopic expression of WOX11 gene driven by the promoter of OsHAK16 encoding a low‐K‐enhanced K transporter led to an extensive root system and adventitious roots and more effective tiller numbers in rice. The WOX11‐regulated root and shoot phenotypes in the OsHAK16p:WOX11 transgenic lines were supported by K‐deficiency‐enhanced expression of several RR genes encoding type‐A cytokinin‐responsive regulators, PIN genes encoding auxin transporters and Aux/IAA genes. In comparison with WT, the transgenic lines showed increases in root biomass, root activity and K concentrations in the whole plants, and higher soluble sugar concentrations in roots particularly under low K supply condition. The improvement of sugar partitioning to the roots by the expression of OsHAK16p:WOX11 was further indicated by increasing the expression of OsSUT1 and OsSUT4 genes in leaf blades and several OsMSTs genes in roots. Expression of OsHAK16p:WOX11 in the rice grown in moderate K‐deficient soil increased total K uptake by 72% and grain yield by 24%–32%. The results suggest that enlarging root growth and development by the expression of WOX11 in roots could provide a useful option for increasing K acquisition efficiency and cereal crop productivity in low K soil.     

Phospholipase Dε enhances Braasca napus growth and seed production in response to nitrogen availability

Phospholipase D (PLD), which hydrolyses phospholipids to produce phosphatidic acid, has been implicated in plant response to macronutrient availability in Arabidopsis. This study investigated the effect of increased PLDε expression on nitrogen utilization in Brassica napus to explore the application of PLDε manipulation to crop improvement. In addition, changes in membrane lipid species in response to nitrogen availability were determined in the oil seed crop. Multiple PLDε over expression (PLDε‐OE) lines displayed enhanced biomass accumulation under nitrogen‐deficient and nitrogen‐replete conditions. PLDε‐OE plants in the field produced more seeds than wild‐type plants but have no impact on seed oil content. Compared with wild‐type plants, PLDε‐OE plants were enhanced in nitrate transporter expression, uptake and reduction, whereas the activity of nitrite reductase was higher under nitrogen‐depleted, but not at nitrogen‐replete conditions. The level of nitrogen altered membrane glycerolipid metabolism, with greater impacts on young than mature leaves. The data indicate increased expression of PLDε has the potential to improve crop plant growth and production under nitrogen‐depleted and nitrogen‐replete conditions.     

Constitutive expression of cell wall invertase genes increases grain yield and starch content in maize

Grain size, number and starch content are important determinants of grain yield and quality. One of the most important biological processes that determine these components is the carbon partitioning during the early grain filling, which requires the function of cell wall invertase. Here, we showed the constitutive expression of cell wall invertase–encoding gene from Arabidopsis, rice (Oryza sativa) or maize (Zea mays), driven by the cauliflower mosaic virus (CaMV) 35S promoter, all increased cell wall invertase activities in different tissues and organs, including leaves and developing seeds, and substantially improved grain yield up to 145.3% in transgenic maize plants as compared to the wild‐type plants, an effect that was reproduced in our 2‐year field trials at different locations. The dramatically increased grain yield is due to the enlarged ears with both enhanced grain size and grain number. Constitutive expression of the invertase‐encoding gene also increased total starch content up to 20% in the transgenic kernels. Our results suggest that cell wall invertase gene can be genetically engineered to improve both grain yield and grain quality in crop plants.     

Lotus SHAGGY‐like kinase 1 is required to suppress nodulation in Lotus japonicus

Glycogen synthase kinase/SHAGGY‐like kinases (SKs) are a highly conserved family of signaling proteins that participate in many developmental, cell‐differentiation, and metabolic signaling pathways in plants and animals. Here, we investigate the involvement of SKs in legume nodulation, a process requiring the integration of multiple signaling pathways. We describe a group of SKs in the model legume Lotus japonicus (LSKs), two of which respond to inoculation with the symbiotic nitrogen‐fixing bacterium Mesorhizobium loti. RNAi knock‐down plants and an insertion mutant for one of these genes, LSK1, display increased nodulation. Ηairy‐root lines overexpressing LSK1 form only marginally fewer mature nodules compared with controls. The expression levels of genes involved in the autoregulation of nodulation (AON) mechanism are affected in LSK1 knock‐down plants at low nitrate levels, both at early and late stages of nodulation. At higher levels of nitrate, these same plants show the opposite expression pattern of AON‐related genes and lose the hypernodulation phenotype. Our findings reveal an additional role for the versatile SK gene family in integrating the signaling pathways governing legume nodulation, and pave the way for further study of their functions in legumes.     

pOsNAR2.1:OsNAR2.1 expression enhances nitrogen uptake efficiency and grain yield in transgenic rice plants

The nitrate () transporter has been selected as an important gene maker in the process of environmental adoption in rice cultivars. In this work, we transferred another native OsNAR2.1 promoter with driving OsNAR2.1 gene into rice plants. The transgenic lines with exogenous pOsNAR2.1:OsNAR2.1 constructs showed enhanced OsNAR2.1 expression level, compared with wild type (WT), and ¹⁵N influx in roots increased 21%–32% in response to 0.2 mm and 2.5 mm and 1.25 mm ¹⁵NH₄¹⁵NO₃. Under these three N conditions, the biomass of the pOsNAR2.1:OsNAR2.1 transgenic lines increased 143%, 129% and 51%, and total N content increased 161%, 242% and 69%, respectively, compared to WT. Furthermore in field experiments we found the grain yield, agricultural nitrogen use efficiency (ANUE), and dry matter transfer of pOsNAR2.1:OsNAR2.1 plants increased by about 21%, 22% and 21%, compared to WT. We also compared the phenotypes of pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines in the field, found that postanthesis N uptake differed significantly between them, and in comparison with the WT. Postanthesis N uptake (PANU) increased approximately 39% and 85%, in the pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines, respectively, possibly because OsNRT2.1 expression was less in the pOsNAR2.1:OsNAR2.1 lines than in the pOsNAR2.1:OsNRT2.1 lines during the late growth stage. These results show that rice NO₃^– uptake, yield and NUE were improved by increased OsNAR2.1 expression via its native promoter.     

Involvement of OsPht1;4 in phosphate acquisition and mobilization facilitates embryo development in rice

Phosphate (Pi) transporters mediate acquisition and transportation of Pi within plants. Here, we investigated the functions of OsPht1;4 (OsPT4), one of the 13 members of the Pht1 family in rice. Quantitative real‐time RT‐PCR analysis revealed strong expression of OsPT4 in roots and embryos, and OsPT4 promoter analysis using reporter genes confirmed these findings. Analysis using rice protoplasts showed that OsPT4 localized to the plasma membrane. OsPT4 complemented a yeast mutant defective in Pi uptake, and also facilitated increased accumulation of Pi in Xenopus oocytes. Further, OsPT4 genetically modified (GM) rice lines were generated by knockout/knockdown or over‐expression of OsPT4. Pi concentrations in roots and shoots were significantly lower and higher in knockout/knockdown and over‐expressing plants, respectively, compared to wild‐type under various Pi regimes. ³³Pi uptake translocation assays corroborated the altered acquisition and mobilization of Pi in OsPT4 GM plants. We also observed effects of altered expression levels of OsPT4 in GM plants on the concentration of Pi, the size of the embryo, and several attributes related to seed development. Overall, our results suggest that OsPT4 encodes a plasma membrane‐localized Pi transporter that facilitates acquisition and mobilization of Pi, and also plays an important role in development of the embryo in rice.     

Chromatin interacting factor OsVIL2 increases biomass and rice grain yield

Grain number is an important agronomic trait. We investigated the roles of chromatin interacting factor Oryza sativa VIN3‐LIKE 2 (OsVIL2), which controls plant biomass and yield in rice. Mutations in OsVIL2 led to shorter plants and fewer grains whereas its overexpression (OX) enhanced biomass production and grain numbers when compared with the wild type. RNA‐sequencing analyses revealed that 1958 genes were up‐regulated and 2096 genes were down‐regulated in the region of active division within the first internodes of OX plants. Chromatin immunoprecipitation analysis showed that, among the downregulated genes, OsVIL2 was directly associated with chromatins in the promoter region of CYTOKININ OXIDASE/DEHYDROGENASE2 (OsCKX2), a gene responsible for cytokinin degradation. Likewise, active cytokinin levels were increased in the OX plants. We conclude that OsVIL2 improves the production of biomass and grain by suppressing OsCKX2 chromatin.     

Gene editing of the wheat homologs of TONNEAU1‐recruiting motif encoding gene affects grain shape and weight in wheat

Grain size and weight are important components of a suite of yield‐related traits in crops. Here, we showed that the CRISPR‐Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1‐recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double‐copy mutant showing larger effect than the respective single copy mutants. The TaGW7‐centered gene co‐expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co‐localization of TaGW7 with α‐ and β‐tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7‐associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR‐Cas9 system with cross‐species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.     

Mutation of the chloroplast‐localized phosphate transporter OsPHT2;1 reduces flavonoid accumulation and UV tolerance in rice

Phosphorus (P) is an essential macronutrient required for plant development and production. The mechanisms regulating phosphate (Pi) uptake are well established, but the function of chloroplast Pi homeostasis is poorly understood in Oryza sativa (rice). PHT2;1 is one of the transporters/translocators mediating Pi import into chloroplasts. In this study, to gain insight into the role of OsPHT2;1‐mediated stroma Pi, we analyzed OsPHT2;1 function in Pi utilization and photoprotection. Our results showed that OsPHT2;1 was induced by Pi starvation and light exposure. Cell‐based assays showed that OsPHT2;1 localized to the chloroplast envelope and functioned as a low‐affinity Pi transporter. The ospht2;1 had reduced Pi accumulation, plant growth and photosynthetic rates. Metabolite profiling revealed that 52.6% of the decreased metabolites in ospht2;1 plants were flavonoids, which was further confirmed by 40% lower content of total flavonoids compared with the wild type. As a consequence, ospht2;1 plants were more sensitive to UV‐B irradiation. Moreover, the content of phenylalanine, the precursor of flavonoids, was also reduced, and was largely associated with the repressed expression of ADT1/MTR1. Furthermore, the ospht2;1 plants showed decreased grain yields at relatively high levels of UV‐B irradiance. In summary, OsPHT2;1 functions as a chloroplast‐localized low‐affinity Pi transporter that mediates UV tolerance and rice yields at different latitudes.     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

Expression of the CCCH‐tandem zinc finger protein gene OsTZF5 under a stress‐inducible promoter mitigates the effect of drought stress on rice grain yield under field conditions

Increasing drought resistance without sacrificing grain yield remains an ongoing challenge in crop improvement. In this study, we report that O ryza s ativa CCCH‐t andem z inc f inger protein 5 (OsTZF5) can confer drought resistance and increase grain yield in transgenic rice plants. Expression of OsTZF5 was induced by abscisic acid, dehydration and cold stress. Upon stress, OsTZF5‐GFP localized to the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF5 under the constitutive maize ubiquitin promoter exhibited improved survival under drought but also growth retardation. By introducing OsTZF5 behind the stress‐responsive OsNAC6 promoter in two commercial upland cultivars, Curinga and NERICA4, we obtained transgenic plants that showed no growth retardation. Moreover, these plants exhibited significantly increased grain yield compared to non‐transgenic cultivars in different confined field drought environments. Physiological analysis indicated that OsTZF5 promoted both drought tolerance and drought avoidance. Collectively, our results provide strong evidence that OsTZF5 is a useful biotechnological tool to minimize yield losses in rice grown under drought conditions.