Laser microdissection transcriptome data derived gene regulatory networks of developing rice endosperm revealed tissue- and stage-specific regulators modulating starch metabolism.

KEY MESSAGE: Laser microdissection applied on the developing rice endosperm revealed tissue- and stage-specific regulators modulating programmed cell death and desiccation tolerance mechanisms in the central starchy endosperm following starch metabolism. Rice (Oryza sativa L.) filial seed tissues are heterozygous in its function, which accumulate distinct storage compounds spatially in starchy endosperm and aleurone. In this study, we identified the 18 tissue- and stage-specific gene co-regulons in the developing endosperm by isolating four fine tissues dorsal aleurone layer (AL), central starchy endosperm (CSE), dorsal starchy endosperm (DSE), and lateral starchy endosperm (LSE) at two developmental stages (7 days after flowering, DAF and 12DAF) using laser microdissection (LM) coupled with gene expression analysis of a 44 K microarray. The derived co-expression regulatory networks depict that distinct set of starch biosynthesis genes expressed preferentially at first in CSE at 7 DAF and extend its spatial expression to LSE and DSE by 12 DAF. Interestingly, along with the peak of starch metabolism we noticed accumulation of transcripts related to phospholipid and glycolipid metabolism in CSE during 12 DAF. The spatial distribution of starch accumulation in distinct zones of starchy endosperm contains specific transcriptional factors and hormonal-regulated genes. Genes related to programmed cell death (PCD) were specifically expressed in CSE at 12DAF, when starch accumulation was already completed in that tissue. The aleurone layer present in the outermost endosperm accumulates transcripts of lipid, tricarboxylic acid metabolism, several transporters, while starch metabolism and PCD is not pronounced. These regulatory cascades are likely to play a critical role in determining the positional fate of cells and offer novel insights into the molecular physiological mechanisms of endosperm development from early to middle storage phase.

Marker-assisted pyramiding of QTLs for heat tolerance and escape upgrades heat resilience in rice (Oryza sativa L.).

KEY MESSAGE: This study demonstrated that pyramiding of early morning flowering and heat tolerance QTLs (qEMF3 and qHTSF4.1) in rice is an efficient approach to maintain high spikelet fertility under high-temperature stress at flowering stage. High temperature at flowering stage of rice causes low spikelet fertility and low yield. To cope with high-temperature stress brought by climate change, two strategies were proposed to develop heat-resilient rice varieties. One is to escape the high temperature by flowering early in the morning, another is to enhance tolerance to high-temperature stress per se. Two promising QTLs for early morning flowering (qEMF3) and heat tolerance (qHTSF4.1) were introgressed into IR64 background, and Near isogenic lines (NILs) IR64 + qEMF3 (IR64EMF3) and IR64 + qHTSF4.1 (IR64HT4) were developed in previous studies. In this study, a QTL pyramiding line IR64 + qHTSF4.1 + qEMF3 (IR64HT4EMF3) was developed by marker-assisted selection of the progenies of previous NILs. The NILs were subjected to different high-temperature regimes in the indoor growth chambers and different locations in the field. In the indoor growth chambers, when high temperature starts early (before 11:00 am), IR64HT4 and IR64HT4EMF3 had higher spikelet fertility than IR64EMF3; when high temperature comes later (after 11:00 am), IR64EMF3 and IR64HT4EMF3 had higher spikelet fertility than IR64HT4. The flowering pattern of the IR64HT4EMF3 was earlier than IR64HT4, but similar to IR64EMF3 in the glasshouse, field and indoor growth chambers. IR64HT4EMF3 showed higher spikelet fertility than IR64EMF3 and IR64HT4 in the field in the Philippines. Thus, combination of early morning flowering and heat tolerance QTLs is an elegant breeding strategy to cope with future extreme climate.

Laser Microdissection-Based Tissue-Specific Transcriptome Analysis Reveals a Novel Regulatory Network of Genes Involved in Heat-Induced Grain Chalk in Rice Endosperm.

Heat stress occurrence during seed filling leads to the formation of a chalky portion in the limited zone of the starchy endosperm of rice grains. In this study, isolation of aleurone, dorsal, central and lateral tissues of developing endosperm by laser-microdissection (LM) coupled with gene expression analysis of a 44 K microarray was performed to identify key regulatory genes involved in the formation of milky-white (MW) and white-back (WB) grains during heat stress. Gene regulatory network analysis classified the genes changed under heat stress into five modules. The most distinct expression pattern was observed in modules where most of the small heat shock proteins and cellular organization genes were changed under heat stress in dorsal aleurone cells and dorsal starchy endosperm zones. The histological observation supported the significant increase in cell number and size of dorsal aleurone cells in WB grains. With regard to the central starchy endosperm zone, preferential down-regulation of high molecular weight heat shock proteins (HMW HSPs), including a prominent member encoding endoplasmic reticulum (ER) chaperones, by heat stress was observed, while changes in expression of starch biosynthesis genes were minimal. Characterization of transgenic plants suppressing endosperm lumenal binding protein gene (BiP1), an ER chaperone preferentially down-regulated at the MW zone under heat stress, showed evidence of forming the chalky grains without disturbing the expression of starch biosynthesis genes. The present LM-based comprehensive expression analysis provides novel inferences that HMW HSPs play an important role in controlling redox, nitrogen and amino acid metabolism in endosperm leading to the formation of MW and WB chalky grains under heat stress.

Fine mapping of a quantitative trait locus for spikelet number per panicle in a new plant type rice and evaluation of a near-isogenic line for grain productivity.

Total spikelet number per panicle (TSN) is one of the determinants of grain productivity in rice (Oryza sativa L.). In this study, we attempted to detect quantitative trait loci (QTLs) for TSN in the introgression lines with high TSN, derived from the cross of Indica Group variety IR 64 with new plant type lines. Two QTLs were detected on the long arm of chromosome 12: qTSN12.1 in the BC4F2 population of YTH63/IR 64 and qTSN12.2 in the BC4F3 population of YTH83/IR 64. TSN of the main tiller was significantly higher in near-isogenic lines (NILs) for qTSN12.1 (IR 64-NIL1; 188.6) and for qTSN12.2 (IR 64-NIL12; 199.4) than in IR 64 (141.2), owing to a significant increase in both primary and secondary branch numbers. These results suggest the critical function of these QTLs in the promotion of rachis branching at the panicle formation stage. Fine mapping of qTSN12.2 revealed six candidate genes in a 92-kb region of the Nipponbare reference genome sequence between flanking markers RM28746 and RM28753. Detailed phenotyping of agronomic traits of IR 64-NIL12 carrying qTSN12.2 showed drastic changes in plant architecture: this line had lower panicle number, longer culm, and longer and wider leaves compared with IR 64. Percentage of fertility and 1000-grain weight tended to be greater, and grain yield per square meter was also greater in IR 64-NIL12 than in IR 64. The newly identified QTLs will be useful for genetic improvement of the yield potential of Indica Group varieties. The markers tightly linked to qTSN12.2 are available for marker-assisted breeding.

NAL1 allele from a rice landrace greatly increases yield in modern indica cultivars.

Increasing crop production is essential for securing the future food supply in developing countries in Asia and Africa as economies and populations grow. However, although the Green Revolution led to increased grain production in the 1960s, no major advances have been made in increasing yield potential in rice since then. In this study, we identified a gene, SPIKELET NUMBER (SPIKE), from a tropical japonica rice landrace that enhances the grain productivity of indica cultivars through pleiotropic effects on plant architecture. Map-based cloning revealed that SPIKE was identical to NARROW LEAF1 (NAL1), which has been reported to control vein pattern in leaf. Phenotypic analyses of a near-isogenic line of a popular indica cultivar, IR64, and overexpressor lines revealed increases in spikelet number, leaf size, root system, and the number of vascular bundles, indicating the enhancement of source size and translocation capacity as well as sink size. The near-isogenic line achieved 13-36% yield increase without any negative effect on grain appearance. Expression analysis revealed that the gene was expressed in all cell types: panicles, leaves, roots, and culms supporting the pleiotropic effects on plant architecture. Furthermore, SPIKE increased grain yield by 18% in the recently released indica cultivar IRRI146, and increased spikelet number in the genetic background of other popular indica cultivars. The use of SPIKE in rice breeding could contribute to food security in indica-growing regions such as South and Southeast Asia.

qEMF3, a novel QTL for the early-morning flowering trait from wild rice, Oryza officinalis, to mitigate heat stress damage at flowering in rice, O. sativa.

A decline in rice (Oryza sativa L.) production caused by heat stress is one of the biggest concerns resulting from future climate change. Rice spikelets are most susceptible to heat stress at flowering. The early-morning flowering (EMF) trait mitigates heat-induced spikelet sterility at the flowering stage by escaping heat stress during the daytime. We attempted to develop near-isogenic lines (NILs) for EMF in the indica-type genetic background by exploiting the EMF locus from wild rice, O. officinalis (CC genome). A stable quantitative trait locus (QTL) for flower opening time (FOT) was detected on chromosome 3. A QTL was designated as qEMF3 and it shifted FOT by 1.5-2.0 h earlier for cv. Nanjing 11 in temperate Japan and cv. IR64 in the Philippine tropics. NILs for EMF mitigated heat-induced spikelet sterility under elevated temperature conditions completing flower opening before reaching 35°C, a general threshold value leading to spikelet sterility. Quantification of FOT of cultivars popular in the tropics and subtropics did not reveal the EMF trait in any of the cultivars tested, suggesting that qEMF3 has the potential to advance FOT of currently popular cultivars to escape heat stress at flowering under future hotter climates. This is the first report to examine rice with the EMF trait through marker-assisted breeding using wild rice as a genetic resource.

Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits.

Flavonoids possess diverse health-promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone-3-hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm-specific GluB-1 promoter or embryo- and aleurone-specific 18-kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB-II-type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes.

Genotypic variations in non-structural carbohydrate and cell-wall components of the stem in rice, sorghum, and sugar vane.

We evaluated genetic variations in the non-structural carbohydrate (NSC) and the cell-wall components of stem in rice, sorghum, and sugar cane to assess the potential suitability of these gramineous crops for bioethanol production. For NSC, the maximum soluble sugar concentration was highest in sugar cane, followed by sorghum with sucrose. The major NSC in rice was starch, but there were wide variations in the starch to soluble sugar ratios among the cultivars. The total concentration of cell-wall components was negatively correlated with the NSC concentration, indicating competition for carbon sources. Among the cell-wall components, lignin was relatively stable within each group. The major sugar species composing hemicellulose was xylose in all crop groups, but there were differences in composition, with a higher fraction of arabinose and glucose in rice as compared to the other crops. In rice, there was less lignin than in sorghum or sugar cane; this might be advantageous for the efficient saccharification of cellulose.

Effect of the light and dark conditions on flower opening time between cultivated rice (Oryza sativa) and a near-isogenic early-morning flowering line.

Flower opening time (FOT) is affected by genetic and environmental factors, but little is known about the effect of light and dark conditions on FOT in cereal crops. FOT of an indica rice cultivar, IR64, and its near-isogenic line carrying a QTL for an early-morning flowering trait (IR64+qEMF3) were investigated in a natural-light and temperature-controlled small greenhouse by exposing either the panicle or stem or both plant organs to different light and dark conditions. FOT did not change in either genotype when panicles were exposed to light. A large difference in FOT was found between genotypes when panicles were exposed to dark conditions; no flower opening was observed in IR64, whereas flower opening was delayed but observed in IR64+qEMF3. These results suggest that the panicle is the organ that perceives light for flower opening in both genotypes, whereas the light requirement to reach flower opening was quite different between genotypes. Flower opening of IR64 occurred concomitantly with elongation of anther filament in the light after the dark treatment stopped, whereas approximately half of flowering of IR64+qEMF3 resulted in apparent cleistogamy even during dark treatment. An extended duration of the dark treatment until 1730H (30-50 min before sunset) made FOT of IR64 spikelets on the next day shifted to a time as early as that of IR64+qEMF3, with significant advancement of FOT compared to the control IR64 spikelets. Our results indicated that different flowering responses to light and dark conditions exist between IR64 and IR64+qEMF3. These findings provide clues for understanding the unique genetic controls of flowering in an EMF line in rice. This study also showed evidence that artificial light environments can shift the FOT of IR64 to that of IR64+qEMF3.

Two Novel QTLs for the Harvest Index that Contribute to High-Yield Production in Rice (Oryza sativa L.).

BACKGROUND: The harvest index (HI) is a measure of the biological success of forming harvestable products. However, our understanding of the genetic basis of HI in rice (Oryza sativa L.) is limited, because it is a complex trait consisting of various yield-related traits and physiological attributes. YTH183 is a high-yielding line with large panicles and high HI derived from a cross between the Indica Group variety IR 64 and the NPT line IR 69093-41-2-3-2 (YP5). RESULTS: Here, we detected two novel QTLs for HI, designated qHI5.1 on chromosome 5 and qHI8.1 on chromosome 8, by using 155 recombinant inbred lines (RILs) derived from the cross between IR 64 and YTH183. The YTH183 allele at qHI5.1 contributed to a wide grain, resulting in heavy grain weight and panicle weight, and was consistently effective under the different environmental conditions of subtropical (Ishigaki) and temperate (Tsukuba) regions. Genetic polymorphism revealed that qHI5.1 was identical to GSE5/GW5, which is known to control the grain weight. On the other hand, although qHI8.1 functioned additively with qHI5.1 for higher HI, it did not show any significant effect on grain or panicle weight. In addition, its effects on HI were shown only in the first seasons at Ishigaki but not at Tsukuba or in the second season at Ishigaki. CONCLUSION: Our results indicate that qHI5.1 controls the grain size, regardless of whether environmental conditions are of subtropical or temperate regions, while qHI8.1 might be involved in controlling the physiological processes of source ability or the translocation of photosynthesis products from vegetative organs to grains depending on environmental conditions during the maturing stage. These QTLs will be useful genetic resources for future breeding programs to break through the ceiling of maximum yield in Indica Group varieties.

The ADAXIALIZED LEAF1 gene functions in leaf and embryonic pattern formation in rice.

The adaxial-abaxial axis in leaf primordia is thought to be established first and is necessary for the expansion of the leaf lamina along the mediolateral axis. To understand axis information in leaf development, we isolated the adaxialized leaf1 (adl1) mutant in rice, which forms abaxially rolled leaves. adl1 leaves are covered with bulliform-like cells, which are normally distributed only on the adaxial surface. An adl1 double mutant with the adaxially snowy leaf mutant, which has albino cells that specifically appear in the abaxial mesophyll tissue, indicated that adl1 leaves show adaxialization in both epidermal and mesophyll tissues. The expression of HD-ZIPIII genes in adl1 mutant increased in mature leaves, but not in the young primordia or the SAM. This indicated that ADL1 may not be directly involved in determining initial leaf polarity, but rather is associated with the maintenance of axis information. ADL1 encodes a plant-specific calpain-like cysteine proteinase orthologous to maize DEFECTIVE KERNEL1. Furthermore, we identified intermediate and strong alleles of the adl1 mutant that generate shootless embryos and globular-arrested embryos with aleurone layer loss, respectively. We propose that ADL1 plays an important role in pattern formation of the leaf and embryo by promoting proper epidermal development.

A genetic resource for early-morning flowering trait of wild rice Oryza officinalis to mitigate high temperature-induced spikelet sterility at anthesis.

BACKGROUND AND AIMS: High temperatures over 32-36 degrees C at anthesis induce spikelet sterility in rice. The use of a germplasm with an early-morning flowering (EMF) trait has been hypothesized as a way of avoiding this problem. In this study, the effect of the EMF trait on avoiding high temperature-induced sterility at anthesis by flowering at a cooler temperature in the early morning was evaluated. METHODS: The EMF trait was introgressed from wild rice (Oryza officinalis) into the rice cultivar 'Koshihikari' (O. sativa). First, spikelets of the EMF line and Koshihikari were subjected to rising temperatures during the daytime in the greenhouse to test for differences in spikelet sterility. Secondly, spikelets of both plants were exposed to 26, 34 and 38 degrees C at anthesis and to 38 degrees C beginning at least 1 h after flowering, in the growth chambers at 70 % relative humidity, to test for differences in tolerance to high temperatures. KEY RESULTS: Spikelets of the EMF line started and completed flowering a few hours earlier than Koshihikari. In a greenhouse experiment, spikelets of Koshihikari opened after the air temperature reached 35 degrees C, but those of the EMF line could open at cooler temperatures. Under these conditions, spikelet sterility significantly increased in Koshihikari, but did not in the EMF line. The number of sterile spikelets increased as their flowering time was delayed in Koshihikari. Furthermore, the chamber experiments revealed that 60 % of the spikelets from both lines were sterile when exposed to 38 degrees C at anthesis, indicating that tolerance of high temperature was similar in both genotypes. CONCLUSIONS: Reduced sterility in the EMF line subjected to rising temperatures at anthesis in the greenhouse was attributed to an earlier flowering time compared with Koshihikari. The EMF trait of wild rice is effective in mitigating anticipated yield loss due to global warming by escaping high-temperature stress at anthesis during the daytime.

The qTSN4 Effect on Flag Leaf Size, Photosynthesis and Panicle Size, Benefits to Plant Grain Production in Rice, Depending on Light Availability.

Increasing rice yield potential is essential to secure world food supply. The quantitative trait locus qTSN4 was reported to achieve yield increases by enhancing both source and sink capacity. Three greenhouse experiments and one field experiment in the Philippines were conducted to study near-isogenic lines (NILs) in two genetic backgrounds, subjected to treatments with restricted light resources through shading (greenhouse) or population density (field and greenhouse). A consistent promotion of flag leaf width, leaf area and panicle size in terms of spikelet number was observed in the presence of qTSN4, regardless of environment. However, grain production per plant was enhanced only in one greenhouse experiment. An in-depth study demonstrated that increased flag leaf size in the presence of qTSN4 was associated with increased photosynthetic rates, along with lower SLA and greater N content per leaf weight and per area. This was emphasized under low light situation as the qTSN4-NILs did not express shade acclimation traits in contrast with the recipient varieties. The authors conclude that qTSN4 is a promising subject for further physiological studies, particularly under limited radiation. However, the QTL alone may not be a reliable source of increased yield potential because its effects at the plant and population scale are prone to genotype × environment interactions and the increased panicle size is compensated by the adaptive plasticity of other morphological traits.

Laser microdissection-based gene expression analysis in the aleurone layer and starchy endosperm of developing rice caryopses in the early storage phase.

BACKGROUND: Rice endosperm is composed of aleurone cells in the outermost layers and starchy endosperm cells in the inner part. The aleurone layer accumulates lipids, whereas starchy endosperm mainly accumulates starch. During the ripening stage, the starch accumulation rate is known to be asynchronous, depending on the position of the starchy endosperm. Different physiological and molecular mechanisms are hypothesized to underlie the qualitative and quantitative differences in storage products among developing rice endosperm tissues. RESULTS: Target cells in aleurone layers and starchy endosperm were isolated by laser microdissection (LM), and RNAs were extracted from each endosperm tissue in the early storage phase. Genes important for carbohydrate metabolism in developing endosperm were analyzed using qRT-PCR, and some of the genes showed specific localization in either tissue of the endosperm. Aleurone layer-specific gene expression of a sucrose transporter, OsSUT1, suggested that the gene functions in sucrose uptake into aleurone cells. The expression levels of ADP-glucose pyrophosphorylase (AGPL2 and AGPS2b) in each endosperm tissue spatially corresponded to the distribution of starch granules differentially observed among endosperm tissues. By contrast, expressions of genes for sucrose cleavage-hexokinase, UDP-glucose pyrophosphorylase, and phosphoglucomutase-were observed in all endosperm tissues tested. Aleurone cells predominantly expressed mRNAs for the TCA cycle and oxidative phosphorylation. This finding was supported by the presence of oxygen (8 % concentration) and large numbers of mitochondria in the aleurone layers. In contrast, oxygen was absent and only a few mitochondria were observed in the starchy endosperm. Genes for carbon fixation and the GS/GOGAT cycle were expressed highly in aleurone cells compared to starchy endosperm. CONCLUSIONS: The transcript level of AGPL2 and AGPS2b encoding ADP-glucose pyrophosphorylase appears to regulate the asynchronous development of starch granules in developing caryopses. Aleurone cells appear to generate, at least partially, ATP via aerobic respiration as observed from specific expression of identified genes and large numbers of mitochondria. The LM-based expression analysis and physiological experiments provide insight into the molecular basis of the spatial and nutritional differences between rice aleurone cells and starchy endosperm cells.

Expression patterns of genes encoding carbohydrate-metabolizing enzymes and their relationship to grain filling in rice (Oryza sativa L.): comparison of caryopses located at different positions in a panicle.

In rice, caryopses located at the base of the panicle have a lower growth rate than those at the tip of the panicle. The former and latter types of caryopses are called inferior and superior caryopses, respectively. Taking the different growth rate into consideration, sugar status and the expression of genes encoding carbohydrate-metabolizing enzymes in inferior caryopses were compared with those in superior caryopses. During the first 5 d after flowering, superior caryopses elongated rapidly, but inferior caryopses did not. At this phase, inferior caryopses had a low ratio of hexose to sucrose, high activity of acid invertase and the absence of the expression of the genes encoding the above enzymes except for two isoforms of cell wall invertase, OsCIN4 and INV1, in comparison with superior caryopses. At the start of caryopsis elongation in both superior and inferior caryopses, the hexose/sucrose ratio increased accompanied by gene expression of vacuolar invertase (INV3), sucrose synthase (RSus1) and ADP-glucose pyrophosphorylase (AGP-L2: D50317). Furthermore, the genes related to endospermal starch accumulation were expressed highly with the decrease in the hexose/sucrose ratio after its peak. Based on the comparison of superior and inferior caryopses, the possible mechanism of grain filling in rice is discussed.

Morphological development of rice caryopses located at the different positions in a panicle from early to middle stage of grain filling.

Rice caryopses show different patterns of grain filling depending on position within a panicle. Caryopses located on the upper primary rachis branches generally accumulate larger amounts of starch at maturity than caryopses located on the secondary rachis branches of the lower primary rachis. In this study, the former and latter types of caryopses were defined as superior and inferior caryopses, respectively. Superior caryopses elongated soon after flowering, whereas inferior caryopses hardly elongated and were morphologically stagnant until the first 4 d after flowering (DAF). However, once inferior caryopses began elongation, their morphological development was the same as superior caryopses until the middle stage of grain filling. Cell division of the inner integument ceased before endosperm cellularization, pericarp functioned as a transient starch storage tissue until endosperm accumulated starch, and endosperm cell number was determined concomitantly with nucellus disintegration. These results implied the coordinated development of the endosperm with maternal tissues. In addition, differences of inner-integument cell number and endosperm cell number were related to a difference of endosperm size between superior and inferior caryopses.