Cell-wall invertases from rice are differentially expressed in Caryopsis during the grain filling stage

Cell-wall Invertase plays an important role in sucrose partitioning between source and sink organs in higher plants. To Investigate the role of cell-wall invertases for seed development In rice (Oryza sativa L.), cDNAs of three putative cell-wall invertase genes OsCIN1, OsCIN2 and OsCIN3 were Isolated. Semi-quantitative reverse transcdption-polymerase chain reaction analysis revealed different expression patterns of the three genes in various rice tissues/organs. In developing ceryopses, they exhibited similar temporal expression patterns, expressed highly at the early and middle grain filling stages and gradually declined to low levels afterward. However, the spatial expression patterns of them were very different, with OsCIN1 primarily expressed in the ceryopsis coat, OsCIN2 in embryo and endosperm, and OsCIN3 In embryo. Further RNA in situ hybridization analysis revealed that a strong signal of OsCIN2 mRNA was detected In the vascular parenchyma surrounding the xylem of the chalazal vein and the aleurone layer, whereas OsCIN3 transcdpt was strongly detected in the vascular parenchyma surrounding the phloem of the chalazal vein, cross.cells, the aleurone layer and the nucellar tissue.These data indicate that the three cell-wall invertase genes play complementary/synergetic roles in assimilate unloading during the grain filling stage. In addition, the cell type-specific expression patterns of OsClN3 In source leaf blades end anthers were also Investigated, and its corresponding physiological roles were discussed.     

Storage-protein Deposition in the Developing Rice Caryopsis in Relation to the Transport Tissues

The developing caryopsis of rice (Oryza sativa L.) was examinedhistologically at successive stages of grain-filling in orderto identify the factors which determine the distribution ofstorage protein in the endosperm, and which terminate the depositionof endosperm protein. The storage protein was deposited at theperiphery of the endosperm, and this distribution was apparentlycaused by the radial pattern of cell development in the endosperm,and by the proximity of the peripheral endosperm cells to thenucellar epidermis. The nucellar epidermis directly surroundsthe endosperm and functions as the pathway for amino acid transportto the endosperm. During the later stages of caryopsis developmentthe nucellar epidermis became compressed by being ‘sandwiched’between the expanding endosperm and the rigid hull (the tightlylocked palea and lemma) which encloses the caryopsis. It isproposed that this compression of the nucellar epidermis blocksthe supply of amino acids to the endosperm and thereby terminatesthe deposition of storage protein in the rice grain. Oryza sativa, rice, caryopsis (development), endosperm, grain filling, nucellar epidermis, storage protein     

Characterization of two members of the maize gene family, Incw3 and Incw4, encoding cell-wall invertases

Two maize putative cell-wall invertase genes (Incw3 and Incw4) have been isolated by screening a genomic DNA library (Zea mays L. W22) using the cDNA probes encoding the two maize cell-wall invertases Incw1 and Incw2. The Incw3 and Incw4 genes contain six exons/five introns and five exons/four introns, respectively. The protein sequences deduced from both genes revealed a beta-fructosidase motif and a cysteine catalytic site known to be conserved in invertase genes. A detailed analysis of the protein and nucleotide sequences provides evidence that the Incw3 and the Incw4 genes encode putative cell-wall invertases. Furthermore, the isoelectric point deduced from the INCW4 protein sequence suggested that the Incw4 gene may encode a unique type of cell-wall invertase unbound in the apoplast. Gene expression studies using RT-PCR and in-situ RT-PCR hybridization showed that the Incw3 expression is organ/tissue-specific and developmentally regulated. In contrast, the Incw4 gene is constitutively expressed in all vegetative and reproductive tissues tested.     

Structural Differentiation of the Nucellar Epidermis in the Caryopsis of Rice (Oryza sativa)

In the developing caryopsis of rice (Oryza sativa L.) the nucellarepidermis forms a uniseriate layer through which assimilatesare transported to the endosperm. An anatomical study demonstratedthat the nucellar epidermal cells are fusiform in shape andare hexagonally packed. The anticlinal walls of the nucellarepidermis are characterized by ribs of wall-thickening whichare orientated radially with respect to the caryopsis. The wall-thickeningsappear to be cellulosic primary walls, as indicated by theirstaining with Calcofluor and periodic acid-Schiff's reagent.It is proposed that the geometry of the nucellar cells and theribs of wall-thickening are structural adaptations to resistthe compressional force which is placed on the nucellar epidermisduring the latter stages of grain filling. Oryza sativa, rice, caryopsis, grain filling, nucellar epidermis, wall-thickening     

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.     

Import of sucrose and its transformation to starch in the developing sorghum caryopsis

Levels of soluble and bound invertases and amylases were studied in relation to the changes in the free sugars and the accumulation of starch in the developing sorghum [Sorghum bicolor (L.) Moench, cv. spv. 351] caryopsis and its associated bractspedicel. Besides sucrose, glucose and fructose as the principal sugars, small amounts of sugars of the raffinose series were detected in the developing caryopsis. Through out the period of caryopsis development, the amount of reducing sugars was higher than that of sucrose. With the advancement in the development of the caryopsis, the contents and levels of sucrose rose with a concomitant fall in the activity of soluble acid (pH 4.8) invertase (EC 3.2.1.26) in the endosperm. In the pericarp-aleurone layer, the activity of soluble acid invertase predominated over soluble neutral (pH 7.5) invertase (EC 3.2.1.27). The activity of bound acid invertase declined with the ageing of the caryopsis. In bracts-pedicel, the activity of bound invertase and the levels of reducing sugars peaked around 18 days post anthesis. In these organs, the level of starch gradually decreased concomitantly with an increase in its level in the developing caryopsis. Amylases (EC 3.2.1.1 and 3.2.1.2) are distributed in the endosperm as well as in the pericarp-aleurone layer. On culturing detached ears in [U-¹⁴C]-sucrose solution for 6 h in the dark at 25°C, 80–90% of the ¹⁴C of extracted major sugars (i.e. sucrose + glucose + fructose) of the caryopsis appeared in sucrose alone. In comparison with the effects of glucose or fructose, transport into the caryopsis of ¹⁴C from [U-¹⁴C]-sucrose supplied to detached ears was promoted by the addition to the radiolabelled sucrose solution of 1% unlabelled sucrose. Addition to the [U-¹⁴C]-sucrose solution fed to the detached ears of 20 mM NaN₃ or HgCl₂ or galactose, lowered the amount of ¹⁴C in the free sugars and starch of the earyopsis.     

Differential regulation of grain sucrose accumulation and metabolism in Coffea arabica (Arabica) and Coffea canephora (Robusta) revealed through gene expression and enzyme activity analysis

* Coffea arabica (Arabica) and Coffea canephora (Robusta) are the two main cultivated species used for coffee bean production. Arabica genotypes generally produce a higher coffee quality than Robusta genotypes. Understanding the genetic basis for sucrose accumulation during coffee grain maturation is an important goal because sucrose is an important coffee flavor precursor. * Nine new Coffea genes encoding sucrose metabolism enzymes have been identified: sucrose phosphate synthase (CcSPS1, CcSPS2), sucrose phosphate phosphatase (CcSP1), cytoplasmic (CaInv3) and cell wall (CcInv4) invertases and four invertase inhibitors (CcInvI1, 2, 3, 4). * Activities and mRNA abundance of the sucrose metabolism enzymes were compared at different developmental stages in Arabica and Robusta grains, characterized by different sucrose contents in mature grain. * It is concluded that Robusta accumulates less sucrose than Arabica for two reasons: Robusta has higher sucrose synthase and acid invertase activities early in grain development - the expression of CcSS1 and CcInv2 appears to be crucial at this stage and Robusta has a lower SPS activity and low CcSPS1 expression at the final stages of grain development and hence has less capacity for sucrose re-synthesis. Regulation of vacuolar invertase CcInv2 activity by invertase inhibitors CcInvI2 and/or CcInvI3 during Arabica grain development is considered.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Soluble invertase expression is an early target of drought stress during the critical,abortion-sensitive phase of young ovary development in maize

To distinguish their roles in early kernel development and stress, expression of soluble (Ivr2) and insoluble (Incw2) acid invertases was analyzed in young ovaries of maize (Zea mays) from 6 d before (-6 d) to 7 d after pollination (+7 d) and in response to perturbation by drought stress treatments. The Ivr2 soluble invertase mRNA was more abundant than the Incw2 mRNA throughout pre- and early post-pollination development (peaking at +3 d). In contrast, Incw2 mRNAs increased only after pollination. Drought repression of the Ivr2 soluble invertase also preceded changes in Incw2, with soluble activity responding before pollination (-4 d). Distinct profiles of Ivr2 and Incw2 mRNAs correlated with respective enzyme activities and indicated separate roles for these invertases during ovary development and stress. In addition, the drought-induced decrease and developmental changes of ovary hexose to sucrose ratio correlated with activity of soluble but not insoluble invertase. Ovary abscisic acid levels were increased by severe drought only at -6 d and did not appear to directly affect Ivr2 expression. In situ analysis showed localized activity and Ivr2 mRNA for soluble invertase at sites of phloem-unloading and expanding maternal tissues (greatest in terminal vascular zones and nearby cells of pericarp, pedicel, and basal nucellus). This early pattern of maternal invertase localization is clearly distinct from the well-characterized association of insoluble invertase with the basal endosperm later in development. This localization, the shifts in endogenous hexose to sucrose environment, and the distinct timing of soluble and insoluble invertase expression during development and stress collectively indicate a key role and critical sensitivity of the Ivr2 soluble invertase gene during the early, abortion-susceptible phase of development.     

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

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