High fructan content in the grain of cereals is an important trait in agriculture such as environmental resilience and dietary fiber food production. To understand the mechanism in

determining final grain fructan content and achieve high fructan cereal, a cross breeding strategy based on fructan synthesis and hydrolysis activities was set up and have achieved barley

lines with 11.8% storage fructan in the harvested grain. Our study discovered that high activity of fructan hydrolysis at later grain developmental stage leads to the low fructan content in

mature seeds, simultaneously increasing fructan synthesis at early stage and decreasing fructan hydrolysis at later stage through crossing breeding is an efficient way to elevate grain

deficiency.

Fructan, a fructose polymer with a terminal sucrose molecule, is produced by many microorganisms, and also by about 15% of plant species1,2,3,4,5. In plants, fructans are produced during

vegetative development and the early stages of reproductive development, which is essential for drought and cold stress tolerance and for grain filling and yield1,2,3,4,5. Some grass

cultivars accumulate high levels of fructans in mature seeds that can be used in a wide range of food and non-food application, e.g., as a functional low-calorie food. Fructan is synthesized

from sucrose in the vacuole of plant cells5. In cereals, the trisaccharide 1-kestotriose is formed by an enzyme named Suc:Suc-1-fructosyltransferase (1-SST, EC 2.4.1.99), which transfers a

fructosyl moiety from one sucrose to the fructose residue of another sucrose. An enzyme called 6G-fructosyl transferase (6G-FFT, EC 2.4.1.243) can use 1-kestotriose as a donor to transfer

the fructosyl moiety to the glucose moiety of sucrose and form neokestose. The trisaccharide 6-kestotriose is formed by a key enzyme named Suc:fructan-6-fructosyltransferase (6-SFT, EC

2.4.1.10) from two sucrose molecules. Further addition of fructosyl moieties to elongate fructosyl chains of fructan from the three trisaccharides as acceptors is catalyzed by the key enzyme

6-SFT, and also by an enzyme called fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100) to form the branched graminan2,3,6,7. Fructan synthesis is also regulated by a transcription

factor called SUSIBA18. Presence of SUSIBA1 inhibits the expression of 6-SFT and blocks fructan synthesis, high level of sucrose could inhibit SUSIBA1 expression and then release 6-SFT to

synthase fructan8.

The mechanism of fructan synthesis has been well understood6,7,8, but until now, it is still difficult to increase reserved fructan content in cereal grain. The function of fructan as a

rapidly available energy-supplying resource during plant growth and development and tolerance and resilience to biotic and abiotic stress and environmental conditions1,2,3,4,9,10 mean that

fructans need to be quickly hydrolyzed to meet these needs. Hydrolysis of fructan in plants is performed by fructan exohydrolases (FEHs) from a terminal of a fructose polymer in the

fructan11. Wheat, and most probably also barley, harbor a set of different FEHs including 1-FEH (fructan 1-exohydrolase, EC 3.3.2.153)11 6-FEH (fructan 6-exohydrolase, EC 3.2.1.154)12,

6&1-FEH13,14 and 6-kestotriose exohydrolase (6-KEH)15. Together, they hydrolyze fructan to sucrose and fructose.

Fructan is generally accepted as a dietary fiber and healthy food ingredient to promote propagation of beneficial bacteria in the digestive systems of mammals and poultry4,16,17.

Consequently, grain fructan has gained high interest in recent years as a functional prebiotic and low-calorie healthy food and feed ingredient2,4,17,18,19. Except fructan,

(1,3;1,4)-β-glucan content in barley grain is also associated with stress tolerance20,21 and both fructan and (1,3;1,4)-beta-glucan content are important in determining barley end

uses2,4,17,18,19. Thus, development of high-fiber cereals is important for both increasing stress tolerance and healthy food production.

As a traditional method, the goal of a cross in inbred breeding is to combine traits presenting in each parent and in some case, achieve transgressive segregation22,23. Thus, progenies have

the possibility to inherit advantage properties from their parents through segregation created by hybridization. The aims of this study were to understand the mechanism in determining final

grain fructan content and to achieve high grain fructan barley with cross breeding strategy by the combination of high fructan synthesis activity and low fructan hydrolysis activity.

In a list of 20 barley lines17, based on the fructan content in mature grain, we selected 12 lines with high, medium, and low levels of fructan content. 249/Gustav, as a commercial variety,

is included as a reference (Supplementary Table S1; Supplementary Fig. S1). During cultivation, we observed that lines 155, 199 accumulated relatively high fructan levels, of over 25% of dry

weight (DW), at an early development stage (9 days after flowering, daf), whereas lines 224 and 235 produced relatively low fructan content at the same stage (Fig. 1a and Supplementary Fig.

S1a). During grain development, the fructan content in all lines decreased gradually to between 0.6 and 3.9% of DW in mature grain (Fig. 1a; Supplementary Fig. S1d). However, we observed

that during the late stages (from 22 daf to grain maturation), the total fructan content in lines 155, and 199 declined more quickly than that in lines 224 and 235 (Fig. 1a). When a diagram

was plotted using total fructan level changes between the two stages, three groups were clearly distinguished (Fig. 1b). Groups 1 and 2 (G1 and G2) showed a relatively large change between

22 daf-mature grains (an over 7% change; Fig. 1b). In contrast, Group 3 (G3) showed a small change during the later stage (around 4% change; Fig. 1b). We hypothesized that it might be

possible to combine the lower reduction in total fructan change at the later stage in G3 with the higher fructan level but also higher fructan reduction at late stage in G1 and G2 by

crossing, to elevate the fructan content. To test this hypothesis, we selected 199, 155, 224 and 235 to perform crossing, that is, 224 (G3) was used as maternal to do cross with 199 (G1) and

155 (G2), and 199 (G1) as maternal to do cross with 235 (G3) (Fig. 1c). During screening process, 20 plants of each crossing line, as F1 generation, were cultivated, and 50 plants of each

crossing line at both F2 and F3 generations were cultivated respectively, to check the segregation and select high fructan plants.

Design of the crossing strategy. (a) Fructan percentage per unit dry weight (DW) during grain development at 9, 15, 22 days after flowering (daf) and at grain maturity for 4 among the 12

barley lines (see Supplementary Table S1 and Supplementary Fig. S1). (b) Percentage change in fructan level between two development stages. Groups 1 (G1), 2 (G2) and 3 (G 3) are indicated by

boxes. (c) Details of the crossing strategy. Pink color indicates maternal and blue color indicates paternal, arrows indicate two crossed varieties. Student’s t-test was used (Error bars

show SD). *P