Molecular, cellular, and developmental foundations of grass diversity.

Humans have cultivated grasses for food, feed, beverages, and construction materials for millennia. Grasses also dominate the landscape in vast parts of the world, where they have adapted morphologically and physiologically, diversifying to form ~12,000 species. Sequences of hundreds of grass genomes show that they are essentially collinear; nonetheless, not all species have the same complement of genes. Here, we focus on the molecular, cellular, and developmental bases of grain yield and dispersal-traits that are essential for domestication. Distinct genes, networks, and pathways were selected in different crop species, reflecting underlying genomic diversity. With increasing genomic resources becoming available in nondomesticated species, we anticipate advances in coming years that illuminate the ecological and economic success of the grasses.

Comparative cellular, physiological and transcriptome analyses reveal the potential easy dehulling mechanism of rice-tartary buckwheat (Fagopyrum Tararicum).

BACKGROUND: Tartary buckwheat has gained popularity in the food marketplace due to its abundant nutrients and high bioactive flavonoid content. However, its difficult dehulling process has severely restricted its food processing industry development. Rice-tartary buckwheat, a rare local variety, is very easily dehulled, but the cellular, physiological and molecular mechanisms responsible for this easy dehulling remains largely unclear. RESULTS: In this study, we integrated analyses of the comparative cellular, physiological, transcriptome, and gene coexpression network to insight into the reason that rice-tartary buckwheat is easy to dehull. Compared to normal tartary buckwheat, rice-tartary buckwheat has significantly brittler and thinner hull, and thinner cell wall in hull sclerenchyma cells. Furthermore, the cellulose, hemicellulose, and lignin contents of rice-tartary buckwheat hull were significantly lower than those in all or part of the tested normal tartary buckwheat cultivars, respectively, and the significant difference in cellulose and hemicellulose contents between rice-tartary buckwheat and normal tartary buckwheat began at 10 days after pollination (DAP). Comparative transcriptome analysis identified a total of 9250 differentially expressed genes (DEGs) between the rice- and normal-tartary buckwheat hulls at four different development stages. Weighted gene coexpression network analysis (WGCNA) of all DEGs identified a key module associated with the formation of the hull difference between rice- and normal-tartary buckwheat. In this specific module, many secondary cell wall (SCW) biosynthesis regulatory and structural genes, which involved in cellulose and hemicellulose biosynthesis, were identified as hub genes and displayed coexpression. These identified hub genes of SCW biosynthesis were significantly lower expression in rice-tartary buckwheat hull than in normal tartary buckwheat at the early hull development stages. Among them, the expression of 17 SCW biosynthesis relative-hub genes were further verified by quantitative real-time polymerase chain reaction (qRT-PCR). CONCLUSIONS: Our results showed that the lower expression of SCW biosynthesis regulatory and structural genes in rice-tartary buckwheat hull in the early development stages contributes to its easy dehulling by reducing the content of cell wall chemical components, which further effects the cell wall thickness of hull sclerenchyma cells, and hull thickness and mechanical strength.

UDP-glucosyltransferase regulates grain size and abiotic stress tolerance associated with metabolic flux redirection in rice.

Grain size is an important component trait of grain yield, which is frequently threatened by abiotic stress. However, little is known about how grain yield and abiotic stress tolerance are regulated. Here, we characterize GSA1, a quantitative trait locus (QTL) regulating grain size and abiotic stress tolerance associated with metabolic flux redirection. GSA1 encodes a UDP-glucosyltransferase, which exhibits glucosyltransferase activity toward flavonoids and monolignols. GSA1 regulates grain size by modulating cell proliferation and expansion, which are regulated by flavonoid-mediated auxin levels and related gene expression. GSA1 is required for the redirection of metabolic flux from lignin biosynthesis to flavonoid biosynthesis under abiotic stress and the accumulation of flavonoid glycosides, which protect rice against abiotic stress. GSA1 overexpression results in larger grains and enhanced abiotic stress tolerance. Our findings provide insights into the regulation of grain size and abiotic stress tolerance associated with metabolic flux redirection and a potential means to improve crops.

An image processing method for investigating the morphology of cereal endosperm cells.

In cereal seeds, the number, morphology and development of endosperm cells are closely related to grain quality, weight and yield. Endosperm cells differ morphologically in different regions of the seed. Nevertheless, it is important to be able to analyze the morphology of cereal endosperm cells. We established an image processing method to enhance the outlines of endosperm cells. The endosperm cell wall was traced precisely using the "pen tool" in Photoshop software (PS). The tracing was defined as the "work path" and was highlighted using the PS "brush tool." Images of mature rice, maize and wheat endosperm sections stained with different methods were analyzed using this method. Combined with the whole sections of mature and developing cereal kernels, the processed image exhibited clearly the morphology of endosperm cells in any region of endosperm and at any stage of endosperm development. The processed image was more accurate and efficient for analyzing morphological characteristics than the unprocessed image.

The Rho-family GTPase OsRac1 controls rice grain size and yield by regulating cell division.

Grain size is a key factor for determining grain yield in crops and is a target trait for both domestication and breeding, yet the mechanisms underlying the regulation of grain size are largely unclear. Here we show that the grain size and yield of rice (Oryza sativa) is positively regulated by ROP GTPase (Rho-like GTPase from plants), a versatile molecular switch modulating plant growth, development, and responses to the environment. Overexpression of rice OsRac1ROP not only increases cell numbers, resulting in a larger spikelet hull, but also accelerates grain filling rate, causing greater grain width and weight. As a result, OsRac1 overexpression improves grain yield in O. sativa by nearly 16%. In contrast, down-regulation or deletion of OsRac1 causes the opposite effects. RNA-seq and cell cycle analyses suggest that OsRac1 promotes cell division. Interestingly, OsRac1 interacts with and regulates the phosphorylation level of OsMAPK6, which is known to regulate cell division and grain size in rice. Thus, our findings suggest OsRac1 modulates rice grain size and yield by influencing cell division. This study provides insights into the molecular mechanisms underlying the control of rice grain size and suggests that OsRac1 could serve as a potential target gene for breeding high-yield crops.

Multi-excitation hyperspectral autofluorescence imaging for the exploration of biological samples.

Many plant tissues can be observed thanks to autofluorescence of their cell wall components. Hyperspectral autofluorescence imaging using confocal microscopy is a fast and efficient way of mapping fluorescent compounds in samples with a high spatial resolution. However a huge spectral overlap is observed between molecular species. As a consequence, a new data analysis approach is needed in order to fully exploit the potential of this spectroscopic technique and extract unbiased chemical information about complex biological samples. The objective of this work is to evaluate multi-excitation hyperspectral autofluorescence imaging to identify biological components in wheat grains during their development through their spectral profiles and corresponding contribution maps using Multivariate Curve Resolution - Alternating Least-Squares (MCR-ALS), a signal unmixing algorithm under proper constraints. For this purpose two different scenarios are used: 1) analyzing the total spectral domain of data sets using MCR-ALS under non negativity constraint in both spectral and spatial modes; 2) analyzing a reduced spectral domain of data sets using MCR-ALS under non negativity in both modes and trilinearity constraint in spectral mode. Considering the original instrumental setup and our data analysis approach, we will demonstrate that extracted contribution maps and spectral profiles of constituents can provide complementary information used to identify molecules in complex biological samples.

Cell Wall Proteome of Wheat Grain Endosperm and Outer Layers at Two Key Stages of Early Development.

The cell wall is an important compartment in grain cells that fulfills both structural and functional roles. It has a dynamic structure that is constantly modified during development and in response to biotic and abiotic stresses. Non-structural cell wall proteins (CWPs) are key players in the remodeling of the cell wall during events that punctuate the plant life. Here, a subcellular and quantitative proteomic approach was carried out to identify CWPs possibly involved in changes in cell wall metabolism at two key stages of wheat grain development: the end of the cellularization step and the beginning of storage accumulation. Endosperm and outer layers of wheat grain were analyzed separately as they have different origins (maternal and seed) and functions in grains. Altogether, 734 proteins with predicted signal peptides were identified (CWPs). Functional annotation of CWPs pointed out a large number of proteins potentially involved in cell wall polysaccharide remodeling. In the grain outer layers, numerous proteins involved in cutin formation or lignin polymerization were found, while an unexpected abundance of proteins annotated as plant invertase/pectin methyl esterase inhibitors were identified in the endosperm. In addition, numerous CWPs were accumulating in the endosperm at the grain filling stage, thus revealing strong metabolic activities in the cell wall during endosperm cell differentiation, while protein accumulation was more intense at the earlier stage of development in outer layers. Altogether, our work gives important information on cell wall metabolism during early grain development in both parts of the grain, namely the endosperm and outer layers. The wheat cell wall proteome is the largest cell wall proteome of a monocot species found so far.

Distribution of cell wall hemicelluloses in the wheat grain endosperm: a 3D perspective.

MAIN CONCLUSION: Uneven distribution of AX and BG in lateral and longitudinal dimensions of a wheat grain was observed by three-dimensional MS imaging, presumably related to specific physicochemical properties of cell walls. Arabinoxylans (AX) and ß-glucans (BG) are the main hemicelluloses that comprise the primary walls of starchy endosperm. These components are not evenly distributed in the endosperm, and the impact of their distribution on cell wall properties is not yet fully understood. Combined with on-tissue enzymatic degradation of the cell walls, mass spectrometry imaging (MSI) was used to monitor the molecular structure of AX and BG in thirty consecutive cross-sections of a mature wheat grain. A 3D image was built from the planar images, showing the distribution of these polymers at the full-grain level, both in lateral and longitudinal dimensions. BGs were more abundant at the vicinity of the germ and in the central cells of the endosperm, while AX, and especially highly substituted AX, were more abundant close to the brush and in the cells surrounding the crease (i.e., the transfer cells). Compared with the previously reported protocol, significant improvements were made in the tissue preparation to better preserve the shape of the fragile sections. This allowed to us achieve a good-quality 3D reconstruction from the consecutive 2D images. By providing a continuous view of the molecular distribution of the cell wall components across and along the grain, the three-dimensional images obtained by MSI may help understand the structure-function relationships of cell walls. The method should be readily extendable to other parietal polymers by selecting the appropriate enzymes.

iTRAQ-Based Proteomics Analysis and Network Integration for Kernel Tissue Development in Maize.

Grain weight is one of the most important yield components and a developmentally complex structure comprised of two major compartments (endosperm and pericarp) in maize (Zea mays L.), however, very little is known concerning the coordinated accumulation of the numerous proteins involved. Herein, we used isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic method to analyze the characteristics of dynamic proteomics for endosperm and pericarp during grain development. Totally, 9539 proteins were identified for both components at four development stages, among which 1401 proteins were non-redundant, 232 proteins were specific in pericarp and 153 proteins were specific in endosperm. A functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the tissue development. Three and 76 proteins involved in 49 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were integrated for the specific endosperm and pericarp proteins, respectively, reflecting their complex metabolic interactions. In addition, four proteins with important functions and different expression levels were chosen for gene cloning and expression analysis. Different concordance between mRNA level and the protein abundance was observed across different proteins, stages, and tissues as in previous research. These results could provide useful message for understanding the developmental mechanisms in grain development in maize.

TaGW2-6A allelic variation contributes to grain size possibly by regulating the expression of cytokinins and starch-related genes in wheat.

MAIN CONCLUSION: Functional allelic variants of TaGW2 - 6A produce large grains, possibly via changes in endosperm cells and dry matter by regulating the expression of cytokinins and starch-related genes via the ubiquitin-proteasome system. In wheat, TaGW2-6A coding region allelic variants are closely related to the grain width and weight, but how this region affects grain development has not been fully elucidated; thus, we explored its influence on grain development based mainly on histological and grain filling analyses. We found that the insertion type (NIL31) TaGW2-6A allelic variants exhibited increases in cell numbers and cell size, thereby resulting in a larger (wider) grain size with an accelerated grain milk filling rate, and increases in grain width and weight. We also found that cytokinin (CK) synthesis genes and key starch biosynthesis enzyme AGPase genes were significantly upregulated in the TaGW2-6A allelic variants, while CK degradation genes and starch biosynthesis-negative regulators were downregulated in the TaGW2-6A allelic variants, which was consistent with the changes in cells and grain filling. Thus, we speculate that TaGW2-6A allelic variants are linked with CK signaling, but they also influence the accumulation of starch by regulating the expression of related genes via the ubiquitin-proteasome system to control the grain size and grain weight.

Short periods of high temperature during meiosis prevent normal meiotic progression and reduce grain number in hexaploid wheat (Triticum aestivum L.).

KEY MESSAGE: Exposure of wheat to high temperatures during male meiosis prevents normal meiotic progression and reduces grain number. We define a temperature-sensitive period and link heat tolerance to chromosome 5D. This study assesses the effects of heat on meiotic progression and grain number in hexaploid wheat (Triticum aestivum L. var. Chinese Spring), defines a heat-sensitive stage and evaluates the role of chromosome 5D in heat tolerance. Plants were exposed to high temperatures (30 or 35 °C) in a controlled environment room for 20-h periods during meiosis and the premeiotic interphase just prior to meiosis. Examination of pollen mother cells (PMCs) from immature anthers immediately before and after heat treatment enabled precise identification of the developmental phases being exposed to heat. A temperature-sensitive period was defined, lasting from premeiotic interphase to late leptotene, during which heat can prevent PMCs from progressing through meiosis. PMCs exposed to 35 °C were less likely to progress than those exposed to 30 °C. Grain number per spike was reduced at 30 °C, and reduced even further at 35 °C. Chinese Spring nullisomic 5D-tetrasomic 5B (N5DT5B) plants, which lack chromosome 5D, were more susceptible to heat during premeiosis-leptotene than Chinese Spring plants with the normal (euploid) chromosome complement. The proportion of plants with PMCs progressing through meiosis after heat treatment was lower for N5DT5B plants than for euploids, but the difference was not significant. However, following exposure to 30 °C, in euploid plants grain number was reduced (though not significantly), whereas in N5DT5B plants the reduction was highly significant. After exposure to 35 °C, the reduction in grain number was highly significant for both genotypes. Implications of these findings for the breeding of thermotolerant wheat are discussed.

Natural Variation in the Promoter of GSE5 Contributes to Grain Size Diversity in Rice.

The utilization of natural genetic variation greatly contributes to improvement of important agronomic traits in crops. Understanding the genetic basis for natural variation of grain size can help breeders develop high-yield rice varieties. In this study, we identify a previously unrecognized gene, named GSE5, in the qSW5/GW5 locus controlling rice grain size by combining the genome-wide association study with functional analyses. GSE5 encodes a plasma membrane-associated protein with IQ domains, which interacts with the rice calmodulin protein, OsCaM1-1. We found that loss of GSE5 function caused wide and heavy grains, while overexpression of GSE5 resulted in narrow grains. We showed that GSE5 regulates grain size predominantly by influencing cell proliferation in spikelet hulls. Three major haplotypes of GSE5 (GSE5, GSE5DEL1+IN1, and GSE5DEL2) in cultivated rice were identified based on the deletion/insertion type in its promoter region. We demonstrated that a 950-bp deletion (DEL1) in indica varieties carrying the GSE5DEL1+IN1 haplotype and a 1212-bp deletion (DEL2) in japonica varieties carrying the GSE5DEL2 haplotype associated with decreased expression of GSE5, resulting in wide grains. Further analyses indicate that wild rice accessions contain all three haplotypes of GSE5, suggesting that the GSE5 haplotypes present in cultivated rice are likely to have originated from different wild rice accessions during rice domestication. Taken together, our results indicate that the previously unrecognized GSE5 gene in the qSW5/GW5 locus, which is widely utilized by rice breeders, controls grain size, and reveal that natural variation in the promoter region of GSE5 contributes to grain size diversity in rice.

The Dynamics of Transcript Abundance during Cellularization of Developing Barley Endosperm.

Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would help predict end-use properties such as yield, quality, and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm 3 d to 8 d after pollination (DAP). Comparisons of transcript abundance over time revealed 47 gene expression modules that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the datasets could be mined to explore and define the processes of cell wall biosynthesis, remodeling, and degradation. Using a combination of spatial molecular network and gene ontology enrichment analyses, it is shown that genes involved in cell wall metabolism are found in multiple modules, but cluster into two main groups that exhibit peak expression at 3 DAP to 4 DAP and 5 DAP to 8 DAP. The presence of transcription factor genes in these modules allowed candidate genes for the control of wall metabolism during early barley grain development to be identified. The data are publicly available through a dedicated web interface (https://ics.hutton.ac.uk/barseed/), where they can be used to interrogate co- and differential expression for any other genes, groups of genes, or transcription factors expressed during early endosperm development.

MicroRNA-targeted transcription factor gene RDD1 promotes nutrient ion uptake and accumulation in rice.

Fertilizers are often potential environmental pollutants, therefore increasing productivity and the efficiency of nutrient uptake to boost crop yields without the risk of environmental pollution is a desirable goal. Here, we show that the transcription factor encoding gene RDD1 plays a role in improving the uptake and accumulation of various nutrient ions in rice. RDD1 was found to be targeted by the microRNA miR166. An RDD1 transgene driven by a strong constitutive promoter exhibited a diurnally oscillating expression similar to that of the endogenous RDD1, and nucleotide substitution within the miR166 recognition site to prevent miR166-RDD1 mRNA pairing resulted in constitutive RDD1 expression. The RDD1 protein was localized to vascular tissue because miR166 repressed RDD1 expression in the mesophyll. The overexpression of RDD1 induced the expression of genes associated with the transport of several nutrients such as NH4(+), Na(+), SO4(2-), Cl(-), PO4(3-) and sucrose, and the uptake and accumulation of various nutrient ions under low-nutrient conditions. Moreover, the overexpression of RDD1 increased nitrogen responsiveness and grain productivity. Our results suggest that RDD1 can contribute to the increased grain productivity of rice via inducing the efficient uptake and accumulation of various nutrient ions.

Metabolic Architecture of the Cereal Grain and Its Relevance to Maximize Carbon Use Efficiency.

Here, we have characterized the spatial heterogeneity of the cereal grain's metabolism and demonstrated how, by integrating a distinct set of metabolic strategies, the grain has evolved to become an almost perfect entity for carbon storage. In vivo imaging revealed light-induced cycles in assimilate supply toward the ear/grain of barley (Hordeum vulgare) and wheat (Triticum aestivum). In silico modeling predicted that, in the two grain storage organs (the endosperm and embryo), the light-induced shift in solute influx does cause adjustment in metabolic flux without changes in pathway utilization patterns. The enveloping, leaf-like pericarp, in contrast, shows major shifts in flux distribution (starch metabolism, photosynthesis, remobilization, and tricarboxylic acid cycle activity) allow to refix 79% of the CO2 released by the endosperm and embryo, allowing the grain to achieve an extraordinary high carbon conversion efficiency of 95%. Shading experiments demonstrated that ears are autonomously able to raise the influx of solutes in response to light, but with little effect on the steady-state levels of metabolites or transcripts or on the pattern of sugar distribution within the grain. The finding suggests the presence of a mechanism(s) able to ensure metabolic homeostasis in the face of short-term environmental fluctuation. The proposed multicomponent modeling approach is informative for predicting the metabolic effects of either an altered level of incident light or a momentary change in the supply of sucrose. It is therefore of potential value for assessing the impact of either breeding and/or biotechnological interventions aimed at increasing grain yield.

The barley anion channel, HvALMT1, has multiple roles in guard cell physiology and grain metabolism.

The barley (Hordeum vulgare) gene HvALMT1 encodes an anion channel in guard cells and in certain root tissues indicating that it may perform multiple roles. The protein localizes to the plasma membrane and facilitates malate efflux from cells when constitutively expressed in barley plants and Xenopus oocytes. This study investigated the function of HvALMT1 further by identifying its tissue-specific expression and by generating and characterizing RNAi lines with reduced HvALMT1 expression. We show that transgenic plants with 18-30% of wild-type HvALMT1 expression had impaired guard cell function. They maintained higher stomatal conductance in low light intensity and lost water more rapidly from excised leaves than the null segregant control plants. Tissue-specific expression of HvALMT1 was investigated in developing grain and during germination using transgenic barley lines expressing the green fluorescent protein (GFP) with the HvALMT1 promoter. We found that HvALMT1 is expressed in the nucellar projection, the aleurone layer and the scutellum of developing barley grain. Malate release measured from isolated aleurone layers prepared from imbibed grain was significantly lower in the RNAi barley plants compared with control plants. These data provide molecular and physiological evidence that HvALMT1 functions in guard cells, in grain development and during germination. We propose that HvALMT1 releases malate and perhaps other anions from guard cells to promote stomatal closure. The likely roles of HvALMT1 during seed development and grain germination are also discussed.

Hormonal requirements for effective induction of microspore embryogenesis in triticale (× Triticosecale Wittm.) anther cultures.

KEY MESSAGE: Effective microspore embryogenesis in triticale is determined by a specific hormonal homeostasis: low value of IAA/cytokinins, IAA/ABA and cytokinins/ABA ratios as well as proper endogenous/exogenous auxin balance, which favours androgenic structure formation and green plant regeneration ability. The concentration of plant growth regulators (PGRs): auxins (Auxs), cytokinins (CKs) and abscisic acid (ABA) was measured in anthers of eight DH lines of triticale (× Triticosecale Wittm.), and associated with microspore embryogenesis (ME) responsiveness. The analysis was conducted on anthers excised from control tillers at the phase optimal for ME induction and then after ME-initiating tillers treatment (21 days at 4 °C). In control, IAA predominated among Auxs (11-39 nmol g(-1) DW), with IBA constituting only 1 % of total Auxs content. The prevailing isoforms of CKs were cis isomers of zeatin (121-424 pmol g(-1) DW) and zeatin ryboside (cZR, 146-432 pmol g(-1) DW). Surprisingly, a relatively high level (10-64 pmol g(-1) DW) of kinetin (KIN) was detected. Cold treatment significantly changed the levels of all analysed PGRs. The anthers of 'responsive' DH lines contained higher concentrations of IBA, cis and trans zeatin, cZR and ABA, and lower amount of IAA and KIN in comparison with 'recalcitrant' genotypes. However, the effects of exogenous ABA, p-chlorophenoxyisobutyric acid (PCIB) and 2,3,5-triiodobenzoic acid treatments suggest that none of the studied PGRs acts alone in the acquisition of embryogenic competency, which seems to be an effect of concerted PGRs crosstalk. The initiation of ME required a certain threshold level of ABA. A crucial prerequisite for high ME effectiveness was a specific PGRs homeostasis: lower Auxs level in comparison with CKs and ABA, and lower CKs/ABA ratio. A proper balance between endogenous Auxs in anthers and exogenous Auxs supplied by culture media was also essential.

Differentiation mechanism and function of the cereal aleurone cells and hormone effects on them.

The cereal aleurone cells differentiate from the endosperm epidermis with the exception of endosperm transfer cells. Aleurone cells contain proteins, lipids, and minerals, and are important for digesting the endosperm storage products to nurse the embryo under effects of several hormones during the seed germination. The differentiation of aleurone cells is related to location effect and special gene expression. Moreover, the differentiation of aleurone cells is probably affected by the cues from maternal tissues. In the paper, differentiation mechanism and function of aleurone cells and hormone effects on them are reviewed. Some speculations about the differentiation mechanism of aleurone cells are given here.

AMT1;1 transgenic rice plants with enhanced NH4(+) permeability show superior growth and higher yield under optimal and suboptimal NH4(+) conditions.

The major source of nitrogen for rice (Oryza sativa L.) is ammonium (NH4(+)). The NH4(+) uptake of roots is mainly governed by membrane transporters, with OsAMT1;1 being a prominent member of the OsAMT1 gene family that is known to be involved in NH4(+) transport in rice plants. However, little is known about its involvement in NH4(+) uptake in rice roots and subsequent effects on NH4(+) assimilation. This study shows that OsAMT1;1 is a constitutively expressed, nitrogen-responsive gene, and its protein product is localized in the plasma membrane. Its expression level is under the control of circadian rhythm. Transgenic rice lines (L-2 and L-3) overexpressing the OsAMT1;1 gene had the same root structure as the wild type (WT). However, they had 2-fold greater NH4(+) permeability than the WT, whereas OsAMT1;1 gene expression was 20-fold higher than in the WT. Analogous to the expression, transgenic lines had a higher NH4(+) content in the shoots and roots than the WT. Direct NH4(+) fluxes in the xylem showed that the transgenic lines had significantly greater uptake rates than the WT. Higher NH4(+) contents also promoted higher expression levels of genes in the nitrogen assimilation pathway, resulting in greater nitrogen assimilates, chlorophyll, starch, sugars, and grain yield in transgenic lines than in the WT under suboptimal and optimal nitrogen conditions. OsAMT1;1 also enhanced overall plant growth, especially under suboptimal NH4(+) levels. These results suggest that OsAMT1;1 has the potential for improving nitrogen use efficiency, plant growth, and grain yield under both suboptimal and optimal nitrogen fertilizer conditions.

SMALL GRAIN 1, which encodes a mitogen-activated protein kinase kinase 4, influences grain size in rice.

Although grain size is one of the most important components of grain yield, little information is known about the mechanisms that determine final grain size in crops. Here we characterize rice small grain1 (smg1) mutants, which exhibit small and light grains, dense and erect panicles and comparatively slightly shorter plants. The short grain and panicle phenotypes of smg1 mutants are caused by a defect in cell proliferation. The smg1 mutations were identified, using a map-based cloning approach, in mitogen-activated protein kinase kinase 4 (OsMKK4). Relatively higher expression of OsMKK4/SMG1 was detected in younger organs than in older ones, consistent with its role in cell proliferation. Green fluorescent protein (GFP)-OsMKK4/SMG1 fusion proteins appear to be distributed ubiquitously in plant cells. Further results revealed that OsMKK4 influenced brassinosteroid (BR) responses and the expression of BR-related genes. Thus, our findings have identified OsMKK4 as a factor for grain size, and suggest a possible link between the MAPK pathways and BRs in grain growth.