Targeting intracellular transport combined with efficient uptake and storage significantly increases grain iron and zinc levels in rice

Rice, a staple food for more than half of the world population, is an important target for iron and zinc biofortification. Current strategies mainly focus on the expression of genes for efficient uptake, long‐distance transport and storage. Targeting intracellular iron mobilization to increase grain iron levels has not been reported. Vacuole is an important cell compartment for iron storage and the NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN (NRAMP) family of transporters export iron from vacuoles to cytosol when needed. We developed transgenic Nipponbare rice lines expressing AtNRAMP3 under the control of the UBIQUITIN or rice embryo/aleurone‐specific 18‐kDa Oleosin (Ole18) promoter together with NICOTIANAMINE SYNTHASE (AtNAS1) and FERRITIN (PvFER), or expressing only AtNRAMP3 and PvFER together. Iron and zinc were increased close to recommended levels in polished grains of the transformed lines, with maximum levels when AtNRAMP3, AtNAS1 and PvFER were expressed together (12.67 μg/g DW iron and 45.60 μg/g DW zinc in polished grains of line NFON16). Similar high iron and zinc levels were obtained in transgenic Indica IR64 lines expressing the AtNRAMP3, AtNAS1 and PvFER cassette (13.65 μg/g DW iron and 48.18 μg/g DW zinc in polished grains of line IR64_1), equalling more than 90% of the recommended iron increase in rice endosperm. Our results demonstrate that targeting intracellular iron stores in combination with iron and zinc transport and endosperm storage is an effective strategy for iron biofortification. The increases achieved in polished IR64 grains are of dietary relevance for human health and a valuable nutrition trait for breeding programmes.     

Molecular characterization of marker-free transgenic lines of <Emphasis Type="Italic">indica</Emphasis> rice that accumulate carotenoids in seed endosperm

A single Agrobacterium strain harbouring two binary plasmids was successfully used for the first time to develop a marker-free transgenic rice of improved nutritional value. Sixty-eight T₀ co-transformants were obtained in three indica rice cultivars—two popular high-yielding Bangladeshi varieties (BR28 and BR29), and one high-iron rice cultivar (IR68144). Marker-free lines were obtained from 14 out of 24 selected co-transformants screened in the T₁ generation. The accumulation of total carotenoids in polished T₂ rice seeds of the primary transgenic VPBR29-17-37 reached levels of up to 3.0 μg/g, with the level of β-carotene reaching 1.8 μg/g. In the cultivars BR28 and IR68144, total carotenoid levels in the transformants reached 2.0 μg/g of polished rice seeds. The levels of lutein and other carotenoids in the seeds were also significantly enhanced. T₁ plants obtained from primary transgenics with simple gene-integration patterns tended to have a lower carotenoid content than the original parental lines. This study describes the development of marker-free transgenic rice lines containing high levels of carotenoids, and addresses the relationship between the rearrangement of transgenes and the presence of metabolic end products in transgenic rice.     

Expression profiling of Oryza sativa metal homeostasis genes in different rice cultivars using a cDNA macroarray.

Rice is an important food crop, but it is a poor source of essential micronutrients such as iron and zinc. In order to improve the metal ion content of rice grains through breeding or biotechnology, more information is needed on the molecular players that help mobilize metals from leaves to developing seeds. To profile several genes simultaneously, a cDNA macroarray was developed using 36 metal-related genes from rice, including ZIPs, NRAMPs, and YSLs (coding for known or potential metal transporters), as well as NAS, FER, FRO, NAAT, FDH, GSTU, and PDR (involved in metal homeostasis). Because flag leaves are the major source of phloem-delivered photoassimilates and remobilized metals for developing seeds, we analyzed the expression of these metal-related genes in flag and non-flag leaves of four different rice cultivars (Cocodrie, Taipei 309, IR58, and IR68144) during the period of mid-grain fill. Genes (24 of 36) exhibited low to non-detectable signals in the macroarray, while 12 genes (OsIRT1, OsZIP1, OsZIP5, OsZIP8, OsYSL5, OsYSL6, OsYSL7, OsYSL8, OsYSL18, OsNRAMP2, OsNRAMP4 and OsNRAMP7) were found to be highly expressed in both flag and non-flag leaves of all four cultivars. Additional expression analysis using semi-quantitative or quantitative PCR provided results that were generally consistent with the macroarray, but semi-quantitative PCR confirmed that OsFDH, OsFER1, OsNAAT, OsNAS1, OsPDR9, OsYSL12, OsYSL13, OsZIP7, and OsZIP10 were also expressed in leaves. This specialized macroarray has provided a short list of potential candidate genes, expressed in leaves, which might contribute to the process of metal transport to distant sinks, such as seeds.     

Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice

Uptake and translocation of micronutrients are essential for plant growth. These micronutrients are also important food components. We generated transgenic rice plants over-expressing OsIRT1 to evaluate its functional roles in metal homeostasis. Those plants showed enhanced tolerance to iron deficiency at the seedling stage. In paddy fields, this over-expression caused plant architecture to be altered. In addition, those plants were sensitive to excess Zn and Cd, indicating that OsIRT1 also transports those metals. As expected, iron and zinc contents were elevated in the shoots, roots and mature seeds of over-expressing plants. This demonstrates that OsIRT1 can be used for enhancing micronutrient levels in rice grains.     

Effect of aluminium on endosperm reserve mobilization in germinating rice grains

The effect of AlCl₃ on endosperm reserve mobilization of rice grains or dehulled rice grains during germination was investigated. AlCl₃ had no effect on grain fresh and dry masses, protein and starch contents, and α-amylase and protease activities in endosperm of germinating rice grains. However, when dehulled rice grains were treated with AlCl₃, AlCl₃ inhibited the decrease in fresh mass, dry mass, and starch and protein contents, and the increase in α-amylase and protease activities in endosperm. Evidence is provided to show that the hull is a barrier against influx of Al to endosperm.This work was supported by the National Science Council of the Republic of China, grant NSC92-2313-B-002-001.     

Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis

Rice ( Oryza sativa L.), the major food staple for more than two billion people, contains neither β-carotene (provitamin A) nor C₄₀ carotenoid precursors thereof in its endosperm. To improve the nutritional value of rice, genetic engineering was chosen as a means to introduce the ability to make β-carotene into rice endosperm tissue. Investigation of the biochemical properties of immature rice endosperm using [¹⁴C]-labelled substrates revealed the presence of geranyl geranyl diphosphate, the C₂₀ general isoprenoid precursor necessary for C₄₀ carotenoid biosynthesis. Phytoene synthase, which condenses two molecules of geranyl geranyl diphosphate, is the first of four specific enzymes necessary for β-carotene biosynthesis in plants. Therefore, the Japonica rice model variety Taipei 309 was transformed by microprojectile bombardment with a cDNA coding for phytoene synthase from daffodil ( Narcissus pseudonarcissus ) under the control of either a constitutive or an endosperm-specific promoter. In transgenic rice plants, the daffodil enzyme is active, as measured by the in vivo accumulation of phytoene in rice endosperm. Thus, it is demonstrated for the first time that it is in principle possible to engineer a critical step in provitamin A biosynthesis in a non-photosynthetic, carotenoid-lacking plant tissue. These results have important implications for longterm prospects of overcoming worldwide vitamin A deficiency.     

The maize disorganized aleurone layer 1 and 2 ( dil1, dil2) mutants lack control of the mitotic division plane in the aleurone layer of developing endosperm

The maize (Zea mays L.) endosperm consists of an epidermal like layer of isodiametric aleurone cells surrounding a central body of starchy endosperm cells. In disorgal1 (dil1) and disorgal2 (dil2) mutants the control of the mitotic division plane is relaxed or missing, resulting in mature grains with disorganized aleurone layers. In addition to orientation of the division plane, both the shape and size of the aleurone cells are affected, and often more than one layer of aleurone cells is present. Homozygous dil1 and dil2 grains are shrunken due to reduced accumulation of starchy endosperm and premature developmental arrest of the embryo, and mature mutant grains germinate at a very low rate and fail to develop into plants. However, homozygous mutant plants can be obtained through embryo rescue, revealing that both mutants have an irregular leaf epidermis as well as roots with a strongly reduced number of root hairs and aberrant root hair morphology. Our results suggest the presence of common regulatory mechanisms for the control of cell division orientation in the aleurone and plant epidermis.Abbreviations DAP days after pollination - dek defective kernel mutant - dil disorganized aleurone layer mutant - GUS -glucuronidase - LM light microscopy - PPB pre-prophase band - SEM scanning electron microscopy - TUSC Trait Utility System for Corn     

Aquaporins in developing rice grains

During rice grain filling, grain moisture content and weight show dynamic changes. We focused on the expression of all 33 rice aquaporins in developing grains. Only two aquaporin genes, OsPIP2;1 and OsTIP3;1, were highly expressed in the period 10–25 days after heading (DAH). High-temperature treatment from 7 to 21 DAH abolished the dynamic up-regulation of OsPIP2;1 in the period 15–20 DAH, whereas OsTIP3;1 expression was not affected. Immunohistochemical analysis revealed that OsPIP2;1 was present in the starchy endosperm, nucellar projection, nucellar epidermis, and dorsal vascular bundles, but not in the aleurone layer. OsTIP3;1 was present in the aleurone layer and starchy endosperm. Water transport activity of recombinant OsTIP3;1 was low, in contrast to the high activity of recombinant OsPIP2;1 we reported previously. Our data suggest that OsPIP2;1 and OsTIP3;1 have distinct roles in developing grains.     

A structural study of germination in celery (Apium graveolens L.) seed with emphasis on endosperm breakdown

Germination of celery seed occurred after 6 d of imbibition in light. During this time the embryo enlarged at the expense of the adjacent endosperm cells and at the time of germination was 2–3 times as long as in the dry seed. Breakdown of the endosperm cells near the root cap preceeded radicle emergence. None of these changes occurred in darkness.Endosperm digestion began adjacent to the embryo and spread radially. In degrading cells, the aleurone grains often became larger and fewer in number. The cell walls were modified and appeared to undergo partial degradation. Ultimately the cells seemed to lose their contents. In cells adjacent to the root cap, similar changes occurred except there was a transient appearance of starch grains. Radial progression of endosperm breakdown also occurred in isolated endosperm treated with gibberellin A₄₊₇.The results indicate that (1) the stimulus for breakdown of celery endosperm emanates from the embryo in response to light; (2) the stimulus may be a gibberellin because changes in endosperm cells and the sequence of endosperm digestion during germination resemble the responses of isolated endosperm to gibberellin; and (3) the radial progression of endosperm breakdown during germination may be the result of a sequential response of cells to a uniformly applied stimulus rather than the result of gradual embryo expansion.     

Posttranslational regulation of pyruvate, orthophosphate dikinase in developing rice (Oryza sativa) seeds

Pyruvate, orthophosphate dikinase (PPDK; E.C.2.7.9.1) is most well known as a photosynthetic enzyme in C₄ plants. The enzyme is also ubiquitous in C₃ plant tissues, although a precise non-photosynthetic C₃ function(s) is yet to be validated, owing largely to its low abundance in most C₃ organs. The single C₃ organ type where PPDK is in high abundance, and, therefore, where its function is most amenable to elucidation, are the developing seeds of graminaceous cereals. In this report, we suggest a non-photosynthetic function for C₃ PPDK by characterizing its abundance and posttranslational regulation in developing Oryza sativa (rice) seeds. Using primarily an immunoblot-based approach, we show that PPDK is a massively expressed protein during the early syncitial-endosperm/-cellularization stage of seed development. As seed development progresses from this early stage, the enzyme undergoes a rapid, posttranslational down-regulation in activity and amount via regulatory threonyl-phosphorylation (PPDK inactivation) and protein degradation. Immunoblot analysis of separated seed tissue fractions (pericarp, embryo + aleurone, seed embryo) revealed that regulatory phosphorylation of PPDK occurs in the non-green seed embryo and green outer pericarp layer, but not in the endosperm + aleurone layer. The modestly abundant pool of inactive PPDK (phosphorylated + dephosphorylated) that was found to persist in mature rice seeds was shown to remain largely unchanged (inactive) upon seed germination, suggesting that PPDK in rice seeds function in developmental rather than in post-developmental processes. These and related observations lead us to postulate a putative function for the enzyme that aligns its PEP to pyruvate-forming reaction with biosynthetic processes that are specific to early cereal seed development.     

ULTRASTRUCTURE OF THE MATURE UNGERMINATED RICE (ORYZA SATIVA) CARYOPSIS. THE CARYOPSIS COAT AND THE ALEURONE CELLS

The results of a light and electron microscopic study of the caryopsis coat and aleurone cells in ungerminated, unimbibed rice (Oryza sativa) caryopses are presented. Surrounding the rice grain is the caryopsis coat composed of the pericarp, seed coat and nucellar layers. The outermost layer, the pericarp, consists of crushed cells and is about 10 μm thick. The seed coat, interior to the pericarp, is one cell thick and has a thick cuticle. Between the seed coat cuticle and endosperm are the remains of the nucellus. The nucellus is about 2.5 μm thick and has a thick cuticle adjacent to the seed coat cuticle. Interior to the caryopsis coat is the aleurone layer of the endosperm. The aleurone completely surrounds the rice grain and is composed of two cell types—aleurone cells that surround the starchy endosperm and modified aleurone cells that surround the germ. The aleurone cells of the starchy endosperm contain many aleurone grains and lipid bodies around a centrally located nucleus. The modified aleurone cells lack aleurone grains, have fewer lipid bodies than the other aleurone cells, and contain filament bundles (fibrils). Plastids of aleurone cells exhibit a unique morphology in which the outer membranes invaginate to form tubules and vesicles within the plastid. Transfer aleurone cells are not observed in the mature rice caryopsis.     

大麦胚和胚乳发育的相关性及贮藏营养物质的积累

大麦(Hordeum vulgare L.)开花后1d,见合子及退化助细胞,游离核胚乳尚未形成;开花后2～3d,胚为5及10个细胞,胚乳为游离核期;开花后4及5、6d,胚为梨形及长梨形,胚乳达细胞化期;开花后8d,胚为胚芽鞘期,糊粉层原始细胞产生;开花后10d,胚具1叶,糊粉层1～2层;开花后13d胚为2叶胚,亚糊粉层发生;开花后17d,3叶胚形成,糊粉层多为3层并停止分裂,菱柱形及不规则胚乳细胞分化;开花后21～29d,胚为4叶胚,胚乳进一步分化;开花后33d,胚为5叶成熟胚,胚乳亦成熟。淀粉、蛋白质在胚中积累始于开花后13d。在盾片中由基向顶发生,在胚芽鞘及叶原基中,首先在顶端出现。成熟盾片顶端的淀粉消失。开花后6d,胚乳开始积累淀粉;开花后10d,糊粉层及胚乳细胞积累蛋白质。开花17d后胚乳的蛋白质体多聚集,29d后蛋白质体显著减少。开花后17d,在盾片及糊粉层细胞中检测到油脂。果长或果长与稃片长之比和盾片长可作为不同发育期胚和胚乳的形态指标。     

Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice

Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa prolamin gene was the only one tested that required both the 5' and 3' flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.     

Overexpression of the Escherichia coli catalase gene, katE, enhances tolerance to salinity stress in the transgenic indica rice cultivar, BR5

Salinity stress is a major limiting factor in cereal productivity. Many studies report improvements in salt tolerance using model plants, such as Arabidopsis thaliana or standard varieties of rice, e.g., the japonica rice cultivar Nipponbare. However, there are few reports on the enhancement of salt tolerance in local rice cultivars. In this work, we used the indica rice (Oryza sativa) cultivar BR5, which is a local cultivar in Bangladesh. To improve salt tolerance in BR5, we introduced the Escherichia coli catalase gene, katE. We integrated the katE gene into BR5 plants using an Agrobacterium tumefaciens-mediated method. The introduced katE gene was actively expressed in the transgenic BR5 rice plants, and catalase activity in T₁ and T₂ transgenic rice was approximately 150% higher than in nontransgenic plants. Under NaCl stress conditions, the transgenic rice plants exhibited high tolerance compared with nontransgenic rice plants. T₂ transgenic plants survived in a 200 mM NaCl solution for 2 weeks, whereas nontransgenic plants were scorched after 4 days soaking in the same NaCl solution. Our results indicate that the katE gene can confer salt tolerance to BR5 rice plants. Enhancement of salt tolerance in a local rice cultivar, such as BR5, will provide a powerful and useful tool for overcoming food shortage problems.