Os04g0413500

GIF1 gene was located on chromosome 4, the exact size of the interval to 32kb. RT-PCR analysis and functional complementation experiments show, Os04g33740 gene that GIF gene, cDNA full length 2089bp, containing the protein product of seven exons encoding 598 amino acids, and gif1 mutant allele occurs one base missing. Analysis showed that GIF is a cell wall converting enzyme protein, with sequence highly similar to corn and tomatoes LIN5 Mn1.

Annotated Information

Function

GIF1 ((grain incomplete filling 1)) plays an important role in crop cultivation which controlling crop grain-filling remain elusive. The cultivated GIF1 gene shows a restricted expression pattern during grain-filling compared to the wild rice allele. Fine mapping with introgression lines revealed that the wild rice GIF1 is responsible for grain weight reduction. The overexpression of GIF1 driven by its native promoter increased grain production. It is suggested that GIF1 is a potential domestication gene and that such a domestication-selected gene can be used for further crop improvement. GIF protein could control rice tissues, including the number of the starch grain. The GIF1 gene in modern cultivated rice is specifically expressed in strict organization which good for grain filling, increasing the yield of rice.The GIF1 gene is responsible for controlling Sucrose Enzyme (enzyme) activity which is located in the cell wall. the GIF1could converse sucrose to starch material for manufacturing.The Sucrose Enzyme play an important role in starch formation. If the Sucrose Enzyme is inactive, rice plants might not make full edible grain. GIF protein is capable of controlling rice tissues, including the number of grains of starch. Modern cultivated GIF1 gene rice has tissues strict specificity, which can in favor of grain filling and improve rice yields.GIF1 gene is responsible for controlling invertase (invertase) activity, wihch is located on the cell wall. Invertase can put sugar into a substance used in the manufacture of starch.GIF1, also encodes a cell-wall invertase that regulates sugar levels in specific tissues, most evidently in the ovular vascular and lateral stylar vascular traces of the developing grain and in the rapidly elongating internodes and roots, where a large amount of sugars is required to support cell division and growth. Assimilated carbon, mainly in the form of sucrose, is transported from the leaf (source) to the vascular trace of seed (sink) . where sucrose is hydrolyzed in the extracellular space into monosaccharides, which are then transported into the endosperm for starch synthesis.GIF1 is a key regulator of this process and has a role in sucrose unloading, which is important in grain development.GIF1was most likely subjected to selection for better grain-filling to achieve a good harvest in cultivated rice. The GIF1gene could increase yield potential through improved grain-filling in the transgenic lines,providing experimental evidence that a domestication-like agronomic trait gene can still be altered by molecular breeding to improve yield in a modern variety.GIF1might be particularly useful for breeding highyield hybrid rice.

Grain-filling and sugar content of gif1 mutant VS. Wild-type rice.

Grain-filling and sugar content of gif1 mutant: The mutant was morphologically normal, with normal seed setting. It showed slower grain-filling than wild-type rice .Consistently,amylose and amylopectin levels were significantly lower in gif1 than in wild-type rice.

Figure 1 Grain-filling and sugar content of gif1 mutant and wild-type rice.(a,b)Grains of gif1 (a) and wild-type (b) rice at 25 DAP (see Supplementary Fig. 1 for seed development). (c,d) White grains of gif1 (c) and wild-type (d)rice. (e,f) Scanning electron microscope analysis of gif1 (e) and wild-type (f)grains. Starch granules developed abnormally and were packed loosely in gif1 grains. Magnification, 1,500. Scale bars represent 10 mm. (g) Grainfilling process (weight in grams of 1,000 brown grains) of gif1 and wild-type (WT) rice. (h–j) Sucrose, glucose and fructose contents of gif1 and wild-type grains. Data in g–j are shown as means ± s.e.m.

Wild-type rice: The gif1 mutant also showed markedly more grain chalkiness as a result of abnormally developed and loosely packed starch granules [.The gif1 mutant also showed markedly more grain chalkiness as a result of abnormally developed and loosely packed starch granules.The reduced filling rate resulted in reduced weight of gif1 grains starting 3 d after pollination (DAP); the final grain weight of the gif1 mutant was B24% lower than that of wild type rice at 30 DAP.

Evolution

GIF1 was paralogues derived from a segmental duplication originated during genome duplication of grasses， was selected in the promoter region.

Expression

1. Wang et al. investigate the site of GIF1 action using a pGIF1- β –glucuronidase (GUS) reporter transgene (pGIF1-GUS). GUS activity was mainly detected in growing roots, the node and the rapidly elongating zone of the internode, similar to its transcript accumulation. Particularly during early grain-filling, strong GUS activity was observed in the ovular vascular trace end of the grain at 3 and 5 DAP. It is proposed that propose that the restricted expression pattern of the GIF1 gene in the ovular vascular trace is the key to increased grain weight. Consistent with our interpretation, transgenic rice plants that ectopically expressed the cultivated GIF1 gene from the rice Waxy promoter also had smaller grains (Figure 2). An extensive microarray survey of gene expression indicates that many genes encoding proteins involved in sugar and energy metabolism have higher expression levels in O. rufipogon than in cultivated rice. The restricted expression pattern of GIF1 in the vascular bundle should facilitate sucrose unloading favoring grain-filling, whereas the wild rice GIF1 allele might promote energy metabolism .

Figure 2 Expression pattern and localization of GIF1. (a,b) GUS activity in the growing root (a) and the node and elongating zone of the internode (b).(c,d) GUS activity in developing grains was restricted to the ovular vascular trace (OV) end at 3 DAP (c) and 5 DAP (d). (e) GUS activity in the OV and lateral stylar vascular trace (SV) of grains at 10 DAP. (f) GUS activity in the OV at 20 DAP. (g) Cross-section of the grain at 10 DAP. (h) Boxed area in g observed under a microscope, showing GUS activity in the OV. NP, nucellar projection. (i) GIF1 transcript levels detected by RT-PCR. UBI1 was used as a loading control. The analysis was repeated twice with similar results. (j) GIF-GFP fusion protein localized to the cell wall in a transgenic rice root tip. Arrow indicates plasmolysis. Scale bars represent 3 mm (c–g) or 100 mm (h).

2. Wang et al. used microarray analysis to examine the regulation of genes involved in sugar metabolis and proposed that sucrose partitioning is altered by the gif1 mutation, leading to lower sucrose levels in the gif1 grain, as similarly reported for carrot .

3.Real-time PCR quantitatively by Wang et al.confirmed expression levels of the wild and cultivated alleles.Similarly elevated expression of wild rice GIF1 was found in other tissues of the introgression lines (data not shown). These results strongly suggest that GIF1 is a domestication gene and that the restricted expression pattern of the cultivated GIF1 gene was caused by nucleotide changes in its promoter during rice domestication .

4. The mapping experiment made by Wang et al. revealed that the wild rice GIF1 is most likely to be the gene that decreases grain weight in the introgression lines. The further analyzed the effect of cultivated alleles on grain-filling using three introgression lines in a recurrent Huajingxian 74 (indica) background with two japonica alleles and one indica allele with distinct origins (in total, two japonica alleles and two indica alleles). It could be proved that these introgression lines showed no difference in grain weight, suggesting that the cultivated rice alleles of indica and japonica have the same effect on grain-filling.

5.GIF1locus on chromosome 4 and then further narrowed it to a 32-kb fragment with three putative genes (Supplementary Fig. 3online). Sequencing of the entire region in the gif1 mutant revealed a 1-nt deletion in the coding region of the Os04g33740gene, causing premature termination of its predicted open reading frame.RT-PCR analysis showed that the Os04g33740transcript level was greatly reduced ingif1grains (Supplementary Fig. 3).TheOs04g33740gene contains seven exons and encodes a protein with 598 amino acids (Supplementary Figs. 3 and 4 online), and its identity as GIF1was verified by functional complementation (Supplementary Fig. 5online) . Database searches indicated that the GIF1 protein is a putative invertase with conserved motifs and a cysteine catalytic site, sharing high sequence similarity with the known maize Mn1 and tomato LIN5 invertases (SupplementaryFig. 4). Invertases constitute a large family in plants;GIF1(also known as OsCIN2) is a member of the cell-wall invertase subfamily with eight members in the rice genome.

Knowledge Extension

1. Grain filling is the process of increasing the weight of every grain of Rice. The grain filling is also the aim which proposed by human. Wang et al. made a research of history of this gene, and proved that its promoter region chose to clean in the process of domestication of rice just as the expectation of people. More researches would concentrate this area which would be helpful in using this gene.

2. There is found that GIF1 and OsCIN1 is a pair of duplication which experienced sub-functionalization implies that selection could act independently on each duplicate towards different functional specificity and provided a vivid example for evolution of genetic novelties in a model crop[1]. Differential biological functions of GIF1 and OsCIN1:

GIF1: GIF1 is mainly expressed in seed vascular tissues and controls sucrose unloading for starch synthesis at the early grain-filling stage. Overexpression of the GIF1 gene produced plants with marked defects both in grainfilling and development, indicating that over-activity of the GIF1 enzyme disrupts sugar homeostasis, a process important to normal grain and plant development.

OsCIN1: OsCIN1 has lower CWI activity compared to GIF1 in the transgenic plants. Consistent with this, no obvious phenotype was observed in CIN1-OE plants except pre-harvest sprouting. Interestingly, OsCIN1 might be involved in pathogen defense and stress response. It has been reported that sugars interact with signaling pathways mediated by phytohormones such as GA and ABA during seed germination and seedling development, which are also involved in stress responses.（Figure 3）

Figure 3 Phenotypes of OsCIN1-OE and GIF1-OE plants. (A) The GIF1-OE plants produced badly-filled grains (right), compared to the empty vector control (left). (B) The OsCIN1-OE plants produced grains completely filled (right). (C) Smaller seeds of the GIF1-OE plants (right), compared to the empty vector control (left). (D) The OsCIN1-OE seeds exhibited preharvest sprouting, while the control seeds did not germinate on panicle at the same time. (E) The GIF1-OE plants grew dwarfing in comparison with the control. (F) The OsCIN1-OE plants were morphologically similar to the control.

3.GIF1 (grain incomplete filling 1) is filling defective mutants screened from flowers 11 mutant strain , with mutant morphology and seed rate on the performance of normal, but the filling degree lower than the wild type, grain starch granules arranged loose, chalky whiteness, the final grain weight lower than 24% of wild-type. Starch, amylopectin and amylose content was significantly lower than that of wild-type.

4.RDesueaprchl iacrtiacletion and independent selection of cell-wall invertase genes GIF1 and OsCIN1 during rice evolution and domestication.

Phylogenetic relationship of cell wall invertases and synteny of the GIF1 and OsCIN1 loci. (A) The N-J phylogenetic tree constructed by MEGA program based on alignment of the DNA sequences of the 8 CWI genes of rice and CWI genes in other species, Lolium perenne, Hordeum vulgare, Dendrocalamopsis oldhamii and the recently released maize and sorghum genomes. Note that the rice GIF1 and OsCIN1 genes were paralogous within two subgroups. (B) Synteny between the GIF1 and OsCIN1 genome regions is illustrated schematically with homologous genes, indicating their duplication event.

Different GIF1 and OsCIN1 expression patterns. (A) Levels of the GIF1 and OsCIN1 transcripts were detected by RT-PCR in different tissues. (B) Levels of the GIF1 and OsCIN1 transcripts during grain filling. Note that OsCIN1 was constitutively expressed in developing grains. The experiments were repeated twice with similar results, Ubi-1 was used as a loading control for RT-PCR. DAP, day after pollination; Cyc, PCR cycles.

5.High yield has been a major breeding target in cereals, including rice(Oryza sativaL.), a staple food crop and a model monocot with thesmallest genome of major cereals. The rice yield trait consists of several key components, including grain number and grain weight,and is regulated by a number of quantitative trait loci (QTLs) derived from natural allelic variations. A few rice genes corresponding to some yield QTLs, such asGn1a, GW2, GS3andGhd7, were recently isolated . The duration and rate of grain-filling, which determine final grain weight and thereby contribute greatly to grain productivity,are also controlled by QTLs. Because of the difficulty in measuring natural variations in grain-filling or weight, map-based cloning of the genes controlling grain-filling has been a major challenge. Wang`s research strongly suggest thatGIF1is a potential domestication gene and that such a domestication-selected gene can be used for further crop improvement.

Labs working on this gene

• Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.

• China National Rice Research Institute, 359 TiyuChang Road, Hangzhou 31006, China.

• Zhejiang Academy of Agricultural Sciences, 198 Shiqiao Road, Hangzhou 310021, China.

• School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China.

• Department of Biology and the Huck Institutes of Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802, USA.

• College of Agriculture, South China Agricultural University, Guangzhou 510642, China.