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.     

Carbon sequestration in soil in a semi‐natural Miscanthus sinensis grassland and Cryptomeria japonica forest plantation in Aso,Kumamoto, Japan

Although Miscanthus sinensis grasslands (Misc‐GL) and Cryptomeria japonica forest plantations (Cryp‐FP) are proposed bioenergy feedstock systems, their relative capacity to sequester C may be an important factor in determining their potential for sustainable bioenergy production. Therefore, our objective was to quantify changes in soil C sequestration 47 years after a Misc‐GL was converted to a Cryp‐FP. The study was conducted on adjacent Misc‐GL and Cryp‐FP located on Mt. Aso, Kumamoto, Japan. After Cryp‐FP establishment, only the Misc‐GL continued to be managed by annual burning every March. Mass C and N, δ¹³C, and δ¹⁵N at 0–30 cm depth were measured in 5 cm increments. Carbon and N concentrations, C:N ratio, δ¹³C, and δ¹⁵N were measured in litter and/or ash, and rhizomes or roots. Although C input in Misc‐GL by M. sinensis was approximately 36% of that in Cryp‐FP by C. japonica, mean annual soil C sequestration in Misc‐GL (503 kg C ha^?1 yr^?1) was higher than that in Cryp‐FP (284 kg C ha^?1 yr^?1). This was likely the result of larger C input from aboveground litter to soil, C‐quality (C:N ratio and lignin concentration in aboveground litter) and possibly more recalcitrant C (charcoal) inputs by annual burning. The difference in soil δ¹⁵N between sites indicated that organic C with N had greater cycling between heterotrophic microbes and soil and produces more recalcitrant humus in Misc‐GL than in Cryp‐FP. Our data indicate that in terms of soil C sequestration, maintenance of Misc‐GL may be more advantageous than conversion to Cryp‐FP in Aso, Japan.     

Allantoin accumulation through overexpression of ureide permease1 improves rice growth under limited nitrogen conditions

In legumes, nitrogen (N) can be stored as ureide allantoin and transported by ureide permease (UPS) from nodules to leaves where it is catabolized to release ammonium and assimilation to amino acids. In non‐leguminous plants especially rice, information on its roles in N metabolism is scarce. Here, we show that OsUPS1 is localized in plasma membranes and are highly expressed in vascular tissues of rice. We further evaluated an activation tagging rice overexpressing OsUPS1 (OsUPS1^OX) under several N regimes. Under normal field conditions, panicles from OsUPS1^OX plants (14 days after flowering (DAF)) showed significant allantoin accumulation. Under hydroponic system at the vegetative stage, plants were exposed to N‐starvation and measured the ammonium in roots after resupplying with ammonium sulphate. OsUPS1^OX plants displayed higher ammonium uptake in roots compared to wild type (WT). When grown under low‐N soil supplemented with different N‐concentrations, OsUPS1^OX exhibited better growth at 50% N showing higher chlorophyll, tiller number and at least 20% increase in shoot and root biomass relative to WT. To further confirm the effects of regulating the expression of OsUPS1, we evaluated whole‐body‐overexpressing plants driven by the GOS2 promoter (OsUPS1^GOS2) as well as silencing plants (OsUPS1^RNAi). We found significant accumulation of allantoin in leaves, stems and roots of OsUPS1^GOS2 while in OsUPS1^RNAi allantoin was significantly accumulated in roots. We propose that OsUPS1 is responsible for allantoin partitioning in rice and its overexpression can support plant growth through accumulation of allantoin in sink tissues which can be utilized when N is limiting.     

Integration of genomic tools to assist breeding in the <Emphasis Type="Italic">japonica</Emphasis> subspecies of rice

The cultivated rice (Oryza sativa L.) has two subspecies, indica and japonica. The japonica rice germplasm has a narrower genetic diversity compared to the indica subspecies. Rice breeders aim to develop new varieties with a higher yield potential, with enhanced resistances to biotic and abiotic stresses, and improved adaptation to environmental changes. In order to face some of these challenges, japonica rice germplasm will have to be diversified and new breeding strategies developed. Indica rice improvement could also profit from more “genepool mingling” for which japonica rice could play an important role. Interesting traits such as low-temperature tolerance, and wider climate adaptation could be introgressed into the indica subspecies. In the past decade, huge developments in rice genomics have expanded our available knowledge on this crop and it is now time to use these technologies for improving and accelerating rice breeding research. With the full sequence of the rice genome, breeders may take advantage of new genes. Also new genes may be discovered from the genepool of wild relatives, or landraces of the genus Oryza, and incorporated into elite japonica cultivars in a kind of “gene revolution” program. Expectedly, new technologies that are currently being optimized, aiming for novel gene discovery or for tracking the regions under selection, will be suggested as new breeding approaches. This paper revisits breeding strategies successfully employed in indica rice, and discusses their application in japonica rice improvement (e.g. ideotype breeding, wide hybridization and hybrid performance).     

A whole‐genome SNP array (RICE6K) for genomic breeding in rice

The advances in genotyping technology provide an opportunity to use genomic tools in crop breeding. As compared to field selections performed in conventional breeding programmes, genomics‐based genotype screen can potentially reduce number of breeding cycles and more precisely integrate target genes for particular traits into an ideal genetic background. We developed a whole‐genome single nucleotide polymorphism (SNP) array, RICE6K, based on Infinium technology, using representative SNPs selected from more than four million SNPs identified from resequencing data of more than 500 rice landraces. RICE6K contains 5102 SNP and insertion–deletion (InDel) markers, about 4500 of which were of high quality in the tested rice lines producing highly repeatable results. Forty‐five functional markers that are located inside 28 characterized genes of important traits can be detected using RICE6K. The SNP markers are evenly distributed on the 12 chromosomes of rice with the average density of 12 SNPs per 1 Mb and can provide information for polymorphisms between indica and japonica subspecies as well as varieties within indica and japonica groups. Application tests of RICE6K showed that the array is suitable for rice germplasm fingerprinting, genotyping bulked segregating pools, seed authenticity check and genetic background selection. These results suggest that RICE6K provides an efficient and reliable genotyping tool for rice genomic breeding.     

Analysis of indica- and japonica-specific markers of Oryza sativa and their applications

Asian rice, Oryza sativa L., is one of the most important crop species. Genetic analysis has established that rice consists of several genetically differentiated variety groups, with two main groups, namely, O. sativa ssp. japonica kata and ssp. indica kata. To determine the genetic diversity of indica and japonica rice, 45 rice varieties, including domesticated rice and Asia common wild rice (O. rufipogon Griff.), were analyzed using sequence-related amplified polymorphism, target region amplified polymorphism, simple sequence repeat, and intersimple sequence repeat marker systems. A total of 90 indica- and japonica-specific bands between typical indica and japonica subspecies were identified, which greatly helped in determining whether domesticated rice is of the indica or japonica type, and in analyzing the consanguinity of hybrid rice with japonica, which were bred from indica and japonica crossed offspring. These specific bands were both located in the coding and non-encoding region, and usually connected with quantitative trait loci. Utilizing the indica-japonica-specific markers, japonica consanguinity was detected in sterile hybrid rice lines. Many indica-japonica-specific bands were found in O. rufipogon. This result supports the multiple-origin model for domesticated rice. Javanica exhibited a greater number of indica-japonica-specific bands, which indicates that it is a subspecies of O. sativa L.     

Humic acid differentially improves nitrate kinetics under low‐ and high‐affinity systems and alters the expression of plasma membrane H+‐ATPases and nitrate transporters in rice

Humic acids (HAs) have a major effect on nutrient uptake, metabolism, growth and development in plants. Here, we evaluated the effect of HA pretreatment applied with a nutrient solution on the uptake kinetics of nitrate nitrogen (N‐NO₃^?) and the metabolism of nitrogen (N) in rice under conditions of high and low NO₃^? supply. In addition, the kinetic parameters of NO₃^? uptake, N metabolites, and nitrate transporters (NRTs) and the plasma membrane (PM) H⁺‐ATPase gene expression were examined. The plants were grown in a growth chamber with modified Hoagland and Arnon solution until 21 days after germination (DAG), and they were then transferred to a solution without N for 48 h and then to another solution without N and with and without the addition of HAs for another 48 h. After this period of N deprivation, the plants received new nutrient solutions containing 0.2 and 2.0 mM N‐NO₃^?. Treatment of rice plants with HA promoted the induction of the genes OsNRT2.1‐2.2/OsNAR2.1 and some isoforms PM H⁺‐ATPase in roots. The application of HAs differentially modified the parameters of the uptake kinetics of NO₃^? under both concentrations. When grown with 0.2 mM NO₃^?, the plants pretreated with HA had lower K_m and C_min values as well as a higher V_max/K_m ratio. When grown with 2 mM NO₃^?, the plants pretreated with HA had a higher V_max value, a greater root and shoot mass, and a lower root/shoot ratio. The N fractions were also altered by pretreatment with HA, and a greater accumulation of NO₃^? and N‐amino was observed in the roots and shoots, respectively, of plants pretreated with HA. The results suggest that pretreatment with HA modifies root morphology and gene expression of PM H⁺‐ATPases and NO₃^? transporters, resulting in a greater efficiency of NO₃^? acquisition by high‐ and low‐affinity systems.     

Using Heading date 1 preponderant alleles from indica cultivars to breed high‐yield,high‐quality japonica rice varieties for cultivation in south China

Heading date 1 (Hd1) is an important gene for the regulation of flowering in rice, but its variation in major cultivated rice varieties, and the effect of this variation on yield and quality, remains unknown. In this study, we selected 123 major rice varieties cultivated in China from 1936 to 2009 to analyse the relationship between the Hd1 alleles and yield‐related traits. Among these varieties, 19 haplotypes were detected in Hd1, including two major haplotypes (H8 and H13) in the japonica group and three major haplotypes (H14, H15 and H16) in the indica group. Analysis of allele frequencies showed that the secondary branch number was the major aimed for Chinese indica breeding. In the five major haplotypes, SNP₃₁₆(C‐T) was the only difference between the two major japonica haplotypes, and SNP₄₉₅(C‐G) and SNP₆₁₄(G‐A) are the major SNPs in the three indica haplotypes. Association analysis showed that H16 is the most preponderant allele in modern cultivated Chinese indica varieties. Backcrossing this allele into the japonica variety Chunjiang06 improved yield without decreasing grain quality. Therefore, our analysis offers a new strategy for utilizing these preponderant alleles to improve yield and quality of japonica varieties for cultivation in the southern areas of China.     

Impact of defoliation intensities on plant biomass,nutrient uptake and arbuscular mycorrhizal symbiosis in Lotus tenuis growing in a saline‐sodic soil

The impact of different defoliation intensities on the ability of Lotus tenuis plants to regrowth, mobilise nutrients and to associate with native AM fungi and Rhizobium in a saline‐sodic soil was investigated. After 70 days, plants were subjected to 0, 25, 50, 75 and 100% defoliation and shoot regrowth was assessed at the end of subsequent 35 days. Compared to non‐defoliated plants, low or moderate defoliation up to 75% did not affect shoot regrowth. However, 100% treatment affected shoot regrowth and the clipped plants were not able to compensate the growth attained by non‐defoliated plants. Root growth was more affected by defoliation than shoot growth. P and N concentrations in shoots and roots increased with increasing defoliation while Na⁺ concentration in shoots of non‐defoliated and moderately defoliated plants was similar. Non‐defoliated and moderately defoliated plants prevented increases of Na⁺ concentration in shoots through both reducing Na⁺ uptake and Na⁺ transport to shoots by accumulating Na⁺ in roots. At high defoliation, the salinity tolerance mechanism is altered and Na⁺ concentration in shoots was higher than in roots. Reduction in the photosynthetic capacity induced by defoliation neither changed the root length colonised by AM fungi nor arbuscular colonisation but decreased the vesicular colonisation. Spore density did not change, but hyphal density and Rhizobium nodules increased with defoliation. The strategy of the AM symbiont consists in investing most of the C resources to preferentially retain arbuscular colonisation as well as inoculum density in the soil.     

Two NHX‐type transporters from Helianthus tuberosus improve the tolerance of rice to salinity and nutrient deficiency stress

The NHX‐type cation/H⁺ transporters in plants have been shown to mediate Na⁺(K⁺)/H⁺ exchange for salinity tolerance and K⁺ homoeostasis. In this study, we identified and characterized two NHX homologues, HtNHX1 and HtNHX2 from an infertile and salinity tolerant species Helianthus tuberosus (cv. Nanyu No. 1). HtNHX1 and HtNHX2 share identical 5′‐ and 3′‐UTR and coding regions, except for a 342‐bp segment encoding 114 amino acids (L₂₇₂ to Q₃₈₅) which is absent in HtNHX2. Both hydroponics and soil culture experiments showed that the expression of HtNHX1 or HtNHX2 improved the rice tolerance to salinity. Expression of HtNHX2, but not HtNHX1, increased rice grain yield, harvest index, total nutrient uptake under K⁺‐limited salt‐stress or general nutrient deficiency conditions. The results provide a novel insight into NHX function in plant mineral nutrition.     

Long‐term biochar application promotes rice productivity by regulating root dynamic development and reducing nitrogen leaching

Biochar is beneficial for improving soil quality and crop productivity. However, the long‐term effects of biochar addition on temporal dynamics of plant shoot and root growth, and the changes in soil properties and nitrogen (N) leaching are still obscure. Here, based on a long‐term (7 years) biochar field experiment with rice in northwest China, we investigated the effects of two biochar rates (0 and 9 t ha^?1 year^?1) and two N fertilizer rates (0 and 300 kg N ha^?1 year^?1) on shoot and root growth, root morphology, N leaching, and soil physicochemical properties. The results showed that both biochar and N fertilizer significantly promoted rice growth, with their interaction significant only in some cases. Both fertilizers enhanced rice shoot biomass and N accumulation in various growth stages as well as increased grain yield. Nitrogen fertilizer significantly promoted root growth regardless of biochar application. However, biochar application without N fertilizer increased root biomass and length during the whole growth period, except in the booting stage; biochar with N application promoted root growth at tillering, reduced root biomass but maintained root length with low root diameter and high specific root length during the jointing and booting stages, and then delayed root senescence in the grain filling stage. Long‐term applications of biochar and N fertilizer reduced 10%–12% bulk density of topsoil compared to the control treatment with no N fertilizer and no biochar. Long‐term biochar application also improved soil total organic carbon and concentrations of available N, phosphorus, and potassium. In addition, biochar and N fertilizer applied together significantly reduced nitrate and ammonium concentration in leachate at different soil depths. In conclusion, biochar could regulate root growth, root morphology, soil properties, and N leaching to increase rice N fertilizer‐use efficiency.     

MutMapPlus identified novel mutant alleles of a rice starch branching enzyme IIb gene for fine‐tuning of cooked rice texture

Physicochemical properties of storage starch largely determine rice grain quality and food characteristics. Therefore, modification of starch property is effective to fine‐tune cooked rice textures. To obtain new resources with modified starch property as breeding materials, we screened a mutant population of a japonica cultivar Nipponbare and found two independent mutant lines, altered gelatinization (age)1 and age2, with moderate changes in starch gelatinization property. A combination of conventional genetic analyses and the latest mapping method, MutMapPlus, revealed that both of these lines harbour novel independent mutant alleles of starch branching enzyme IIb (BEIIb) gene. In age1, amino acid substitution of Met‐723 to Lys completely abolished BEIIb enzyme activity without significant reduction in its protein level. A transposon insertion in an intron of BEIIb gene reduced BEIIb protein level and activity in age2. Production of a series of the mutant lines by combining age alleles and indica‐type starch synthase IIa allele established stepwise alteration of the physicochemical properties of starch including apparent amylose content, thermal property, digestibility by α‐amylase and branched structures of amylopectin. Consistent with the alteration of starch properties, the results of a sensory evaluation test demonstrated that warm cooked rice of the mutants showed a variety of textures without marked reduction in overall palatability. These results suggest that a series of the mutant lines are capable of manipulation of cooked rice textures.     

Single-seeded InDel fingerprints in rice:An effective tool for indica-japonica rice classification and evolutionary studies

Asian cultivated rice(Oryza sativa L.),an important cereal crop worldwide,was domesticated from its wild ancestor 8000 years ago.During its long-term cultivation and evolution under diverse agroecological conditions, Asian cultivated rice has differentiated into indica and japonica subspecies.An effective method is required to identify rice germplasm for its indica and japonica features,which is essential in rice genetic improvements.We developed a protocol that combined DNA extraction from a single rice seed and the insertion/deletion(InDel) molecular fingerprint to determine the indica and japonica features of rice germplasm.We analyzed a set of rice germplasm,including 166 Asian rice varieties,two African rice varieties,30 accessions of wild rice species,and 42 weedy rice accessions,using the single-seeded InDel fingerprints(SSIF).The results show that the SSIF method can efficiently determine the indica and japonica features of the rice germplasm.Further analyses revealed significant indica and japonica differentiation in most Asian rice varieties and weedy rice accessions.In contrast,African rice varieties and nearly all the wild rice accessions did not exhibit such differentiation.The pattern of cultivated and wild rice samples illustrated by the SSIF supports our previous hypothesis that indica and japonica differentiation occurred after rice domestication under different agroecological conditions.In addition,the divergent pattern of rice cultivars and weedy rice accessions suggests the possibility of an endoferal origin(from crop)of the weedy rice included in the present study.