High-quality sugar production by osgcs1 rice

Abstract

Carbohydrates (sugars) are an essential energy-source for all life forms. They take a significant share of our daily consumption and are used for biofuel production as well. However, sugarcane and sugar beet are the only two crop plants which are used to produce sugar in significant amounts. Here, we have discovered and fine-tuned a phenomenon in rice which leads them to produce sugary-grain. We knocked-out GCS1 genes in rice by using CRISPR technology, which led to fertilization failure and pollen tube-dependent ovule enlargement morphology (POEM) phenomenon. Apparently, the POEMed-like rice ovule ('endosperm-focused') can grow near-normal seed-size unlike earlier observations in Arabidopsis in which gcs1 ovules ('embryo-focused') were aborted quite early. The POEMed-like rice ovules contained 10-20% sugar, with extremely high sucrose content (98%). Trancriptomic analysis revealed that the osgcs1 ovules had downregulation of starch biosynthetic genes, which would otherwise have converted sucrose to starch. Overall, this study shows that pollen tube content release is sufficient to trigger sucrose unloading at rice ovules. However, successful fertilization is indispensable to trigger sucrose-starch conversion. These findings are expected to pave the way for developing novel sugar producing crops suited for diverse climatic regions.

Fig. 1. OsGCS1 and OsGCS1-like genes and phenotypes of their respective mutants.

a OsGCS1 (Os05g0269500) and OsGCS1-like genes (Os09g0525700) with the mutation sites (g28, g83, g51, and g41 with red lightning). Vertical bars (exons) and horizontal lines (introns). b Tissue-specific qRT-PCR expression analysis of OsGCS1 and OsGCS1-like genes. OsACT1 (Os03g50885) was used as an internal control. The bar whiskers represent SEM of two biological and six technical replicates (**p < 0.001 in two-tailed t-test). c, d Aniline blue staining. Only one pollen tube (PT) can be seen which is inserted to micropyle (MP) in Nipponbare ovary (1DAP) (n = 24 pistils) (c). In osgcs1 (g51) mutant (1DAP), PT1 has inserted to micropyle, however, PT2 has not yet (n = 68) (d). osgcs1 PTs had no PT guidance defects as in Arabidopsis. Bars: 100 μm. e, f A Nipponbare grain. A border of the embryo (Eb) and endosperm (Es) (Dashed line). Pc pericarp, Tm tegmen, and AL aleurone layer. g, h osgcs1 grain lacks embryo, endosperm and aleurone layer but transparent liquid (*). f, h Toluidine blue staining. Bars: 1 mm. i Nipponbare seed and an osgcs1-like (Tos) mutant seed-like structure with reminiscent of a stigma (St) (n = 10). j Transparent liquid (TL) from the structure. k Watery seed-like tissue production by OsGCS1 or OsGCS1-like gene mutations. Each box represents the data at 25th percentile (lower end), median (horizontal line within the box), and the data at 75th percentile (upper end). The outer ends of whiskers at each box represent the maximum and minimum data points. Outliers, when present, are represented by small circle with associated data value (*p ≤ 0.5, **p ≤ 0.05, Tukey-Kramer multiple comparison test; n = 10 (NB Nipponbare), 10 (EV empty vector), 5 (g28), 5 (g83), 6 (g51), 4 (Tos), and 5 (g41)).

Fig. 2. Typical gene expression pattern in the osgcs1 mutant with high sucrose accumulation in its ovules.

a Hierarchical clustering of transcriptome data obtained from Nipponbare (NB) ovules before pollination (NB 0DAP), NB ovules at 1 and 3 days after pollination (NB 1DAP and NP 3DAP), and osgcs1 ovules at 1 and 3 days after pollination (osgcs1 1DAP and osgcs1 3DAP). b Early-response genes triggered by PTC release were screened in the two classes indicated and pooled for further expression analyses in c (see “Method” for details). c Temporal expression of the identified genes in NB and osgcs1 ovules. d Temporal expression of the genes for cell expansion (expansin), cell division (cyclin), and starch synthesis in NB and osgcs1 ovules. e Metabolic cascade for starch synthesis from sucrose in rice. The expression of genes encoding the enzymes generating glucose or fructose from sucrose and the enzymes acting at steps further downstream for starch production were down-regulated in osgcs1 ovules compared to NB ovules (marked in blue triangles). f Sugar component promotion of the fluid from osgcs1 ovules. The fluid contained 1% glucose, 1% fructose, and 98% sucrose. The bar whiskers represent SEM of respective lines (**p ≤ 0.05, Tukey’s test; n = 5). g Sugar content of the fluid obtained from osgcs1 ovules. Each box represents the data at 25th percentile (lower end), median (horizontal line within the box), and the data at 75th percentile (upper end). The outer ends of whiskers at each box represent the maximum and minimum data points (**p ≤ 0.05, Tukey’s test; n = 5).

Fig. 3. Proposed mechanism of sugar rice production.

Sucrose synthesized in rice leaves after photosynthesis is loaded to phloem. Its unloading into ovules should be triggered by pollen tube content (PTC) release. While successful fertilization after PTC release further triggers sucrose–starch conversion and development of normal starchy rice grains, the fertilization defect in osgcs1 ovules should lead to the continued accumulation of sucrose and development of sugar rice. In absence of pollen tube or PTC reception, ovules cannot sustain their prolonged growth and are subsequently aborted. The purple square in the typical rice ovule is expanded to show sucrose unloading and/or sucrose–starch conversion in subsequent steps, while the red square has been expanded to show fertilization steps. The path of sugar rice development has been highlighted in light navy blue color. ph phloem; c companion cell; nu nucellus; integ integuments (inner and outer); AC antipodal cells; CC central cell; SC synergid cell; EC egg cell; pn polar nuclei; vb vascular bundle; o ovule.