Source-Sink and Grain-Filling Characteristics of Two-Line Hybrid Rice Yangliangyou 6

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1 34 Rice Science, 2006, 13(1): Source-Sink and Grain-Filling Characteristics of Two-Line Hybrid Rice Yangliangyou 6 ZHAO Bu-hong 1,2, WANG Peng 1, ZHANG Hong-xi 2, ZHU Qing-sen 1, YANG Jian-chang 1 ( 1 Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Yangzhou University, Yangzhou , China; 2 Lixiahe Region Agricultural Research Institute of Jiangsu, Yangzhou , China) Abstract: With two-line hybrid rice Yangliangyou 6 (YLY6) and Liangyoupeijiu (LYPJ) and three-line hybrid rice Shanyou 63 (SY63) as materials, the source, sink and flow characteristics in association with grain filling were investigated. The seed-setting rate, grain filling degree and grain yield of YLY6 and SY63 were significantly higher than those of LYPJ. The export and transformation percentages of the matter in culms and sheaths of YLY6 and SY63 were significantly higher than those of LYPJ. Activities of sucrose synthase, adenosine diphosphoglucose pyrophosphorylase, starch synthase and starch branching enzyme in grains were higher for YLY6 and SY63 than for LYPJ, and were very significantly correlated with maximum grain filling rate, mean grain filling rate, grain filling degree and grain weight. The spikelet number, grain yield and total sink load per area of vascular bundle and phloem of YLY6 and SY63 were significantly smaller than those of LYPJ, and the greater the load, the lower seed-setting rate and the poorer grain filling. The transportation rate per area phloem of YLY6 was greater than that of LYPJ or SY63. The results suggest that YLY6 possesses strong source, great sink activity and efficient flow, which lay a physiological base for its high seed-setting rate and good grain filling. Key words: hybrid rice; source-sink characteristics; grain filling; enzyme activity Two-line hybrid rice combinations have obvious super high-yielding characteristics of strong matter productivity and large sink size. However, their lower seed setting rate, higher empty grain rate and unfilled grain rate, and poor grain filling degree, seriously limit their yield potential [1-3]. Some studies have been done on characteristics of matter productivity, grain filling, and matter translocation, etc, to explain the reasons for lower seed setting rate and poor grain filling degree in two-line hybrid rice, but the results were controversial [4-6]. Yangliangyou 6 (YLY6), a new two-line hybrid rice combination, was bred in Lixiahe Region Agricultural Research Institute of Jiangsu Province during the past years. This combination has a characteristic of high yield, good quality and strong resistance to diseases. However, little is known about its characteristics of grain-filling and source-sink so far. In this study, characteristics of source-sink and flow and their relationships with grain filling of YLY6 Received: 14 November 2005; Accepted: 23 January 2006 Corresponding author: YANG Jian-chang (jcyang@yzu.edu.cn) were comprehensively analyzed in respects of matter translocation, sink activity, and vascular bundle. The purposes were to put forward theoretical and practical guidance for high-yielding and good quality in super two-line hybrid rice breeding and cultivation. MATERIALS AND METHODS Materials and cultivation Two two-line hybrid rice combinations YLY6 and LYPJ, and one three-line hybrid rice combination SY63, were grown in the Farm of Yangzhou University, Yangzhou, China in 2002 and Seeds were sown on 5-10 May and the seedlings were transplanted on 5-10 June with one seedling per 15 cm 25 cm area. The plot area was 18.0 m 2 (3.0 m 6.0 m). A completely randomized block design with three replications was used in the study. Total nitrogen fertilizer applied during the whole growth period was 420 kg urea per hectare according to the ratio of 6:1:3 at basal, tillering, and panicle initiation stages. The

2 ZHAO Bu-hong, et al. Source-Sink and Grain-Filling Characteristics of Two-Line Hybrid Rice Yangliangyou 6 35 average temperatures from heading to harvest in every 5 days were 27.5, 27.1, 26.6, 25.3, 24.5, 23.6, 22.5, 22.1, 21.2 and 20.7, respectively, and which were suitable for grain filling. Sampling and measurements Number and area of vascular bundle Twenty panicles headed on the same day were chosen and culms were sampled at 0.5 cm under panicle node. The methods for determining number and area of vascular bundle were modified from Huang et al [4]. Dry matter Plants of 10 hills from each plot were sampled at the heading and maturity, and they were separated into leaves, culms and sheaths, and panicles. Dry matter of each component was determined after drying at 70 to constant weight. The export percentage of the matter in culms and sheaths (EPMCS) and transformation percentage of the matter in culms and sheaths (TPMCS) were calculated according to the following equations: EPMCS(%) = (dry matter weight in culms and sheaths at heading-dry matter weight in culms and sheaths at maturity)/ dry matter weight in culms and sheaths at heading 100%. TPMCS(%) = (dry matter weight in culms and sheaths at heading-dry matter weight in culms and sheaths at maturity)/ panicle weight at maturity 100%. Enzyme activities Two hundred to 250 panicles headed on the same day were chosen and tagged in each plot. The flowering date and position were recorded. Eight to 10 tagged panicles from each plot were sampled at a 3-day interval from flowering to 21 days after flowering (DAF), and 6-day interval from 21 DAF to maturity. Superior grains (flowered on the 1st and 2nd day on the panicle) and inferior grains (flowered on the last three days on the panicle) were taken off from sampled panicles. Half-sampled grains were frozen in liquid nitrogen for 1 min then stored at -70 for enzymatic measurement. Another half grains were dried at 70 to constant weight, dehulled and weighed for grain growth analysis and starch content measurement. The methods for sucrose synthase (SuSase), adenosine diphosphoglucose pyrophosphorylase (AGPase), starch synthase (StSase), starch branching enzyme (BE) and acid invertase (AIV) were modified from Nakamura et al [5]. The grain-filling process was fitted by Richards growth equation as described by Zhu et al [7-8]. Yield and its components Plants of 10 hills from each plot were harvested at maturity to determine the yield components. The grain filling percentage was determined by Zhu et al [8]. Plants from a 5-m 2 area in each plot were harvested at maturity for the determination of grain yield. RESULTS Grain yield and its components The grain yield and grain-filling characteristics of the combinations were shown in Table 1. The grain yield of YLY6 was 8875 kg/ha, significantly higher than those of the other two combinations, which was mainly due to its greater number of spikelets, higher seed setting rate and filling degree. The grain yield of LYPJ was only 7183 kg/ha, significantly lower than the other two combinations, which was mainly associated with its lower seed setting rate and filling degree (Table 1). Table 1. Grain yield and its components. No. of panicles ( 10 4 /ha) No. of spikelets per panicle Seed setting rate (%) Empty grain rate (%) Unfilled grain rate (%) Degree of grain filling (%) 1000-grain weight (g) Grain yield (kg/ha) Liangyoupeijiu b a b a a b c 7183 c Yangliangyou b a a 5.95 b b a a 8875 a Shanyou a b a 5.02 b b a b 8081 b Within a column, data followed by the same lowercase letters indicated no significance at 0.05 level.

3 36 Rice Science, Vol. 13, No. 1, 2006 Characteristics of source-sink and grain filling Different types of grain-leaf ratio (leaf area was measured at the heading stage) reflecting source-sink characteristics were listed in Table 2. The significant differences of source-sink characteristics in the same combination were observed when using different types of grain-leaf ratio. The coefficients of variation of yield sink per unit area and actual yield per unit area was much lower, indicating they could reflect the real characteristics of source-sink among different combinations, because they partly eliminated the interaction between spikelet number and grain weight. Grain filling index (seed setting rate filling degree) comprehensively reflected the status of coordination between source and sink. Grain filling indices of SY63 and YLY6 were higher, indicating that the relation between source and sink was basically concordant. Grain filling index was lower for LYPJ, indicating that source use efficiency is lower or source potential is inefficient, which was probably related to lower matter production after heading, or lower matter translocation efficiency during the late growth period. As a whole, the yield sink per unit leaf area of LYPJ and YLY6 was higher than that of SY63. Matter translocation and grain filling There were no significant differences in dry matter accumulation at the heading stage among the tested combinations (Table 3). Dry matter accumulation of LYPJ and YLY6 after heading was significantly higher than that of SY63. At maturity, dry matter weight in order was YLY6 > LYPJ > SY63, indicating that two-line hybrid rice has superiority in matter production during the grain filling period. The export and transformation percentages of the matter in culms and sheaths showed SY63 > YLY6 > LYPJ in order. The export and transformation percentages of the matter in culms and sheaths of YLY6 were significantly higher than those of LYPJ, but were not significantly different with those of SY63 (Table 3). The results suggested that matter translocation characteristics were significantly different among two-line hybrid rice combinations, and the higher the seed setting rate and grain filling degree, the higher the matter translocation rate. Significant differences were found among three combinations in grain-filling rate and grain-filling period (Table 4). SY63 had the shortest time to reach maximum grain-filling rate and maximum grain weight and its mean grain-filling rate was the highest. LYPJ had a longer time than YLY6 to reach maximum grain-filling rate and maximum grain weight, but its mean grain-filling rate was significantly lower than that of YLY6. Table 2. Comparison of source-sink ratio among various hybrid rice combinations. No. of spikelets per 1 cm 2 leaf No. of grains per 1 cm 2 leaf Ratio of grain yield to leaf area (mg/cm 2 ) Ratio of yield sink to leaf area (mg/cm 2 ) Filling index (%) Liangyoupeijiu 0.81 a 0.62 b c a b Yangliangyou a 0.70 a a a a Shanyou b 0.61 b b b a Average CV (%) Data followed by the different letters indicated significant difference at 0.05 level among the combinations. Table 3. Dry matter (DW) accumulation and transformation of different hybrid rice combinations. DW at heading DW at maturity DW after heading Panicle weight EPMCS TPMCS (g/m 2 ) (g/m 2 ) (g/m 2 ) (g/m 2 ) (%) (%) Liangyoupeijiu a a a c b b Yangliangyou a a a a a a Shanyou a b b b a a EPMSS, Export percentage of the matter in culms and sheaths; TPMSS, Transformation percentage of the matter in culms and sheaths. Within a column, data followed by the different letters indicated significant difference at 0.05 level.

4 ZHAO Bu-hong, et al. Source-Sink and Grain-Filling Characteristics of Two-Line Hybrid Rice Yangliangyou 6 37 Table 4. Parameters of grain-filling characteristics. Time reaching maximum grain-filling rate (d) Maximum grain-filling rate (mg/d grain) Mean grain-filling rate (mg/d grain) Initial growth power (R o) Time reaching maximum grain weight (d) Liangyoupeijiu a b b b a Yangliangyou b a a a b Shanyou c a a a c Within a column, data followed by the same lowercase letters indicated no significant difference at 0.05 level. Vascular bundle characteristics and grain filling YLY6 was significantly higher than either SY63 or LYPJ in terms of the number of vascular bundles of peduncle, the individual area of phloem and xylem, and the total area of phloem and xylem. There were no significant differences between SY63 and LYPJ in the characteristics of vascular bundles of peduncle (Table 5). The vascular bundles, spikelets, seed setting rate and total sink size per unit phloem area were low for the combinations with good grain filling (SY63 and YLY6), and were high for the combination with poor grain filling (LYPJ) (Table 6). Matter translocation rate per unit phloem area was higher for the combination with good grain filling than that with poor grain filling, indicating that high transportation rate per unit phloem area is beneficial to grain filling. Starch accumulation in grains Changes of starch content in grains in early filling period were as follows: Starch accumulation in superior grains was higher, being about 38 40% of the maximum grain weight on 4 days after flowering (DAF), about 80% on 16 DAF and 80 85% on 20 DAF. From 20 DAF to maturity, it changed slightly. Starch content in inferior grains was rather lower at the early grain filling stage, only about 15 20% of the maximum grain weight on 4 DAF, and about 40 60% on 16 DAF. On 32 DAF, starch content in inferior grain was close to that in superior grains (Fig. 1, A-C). Changes of soluble sugar content in grains were contrary to those of starch content. At early and mid grain filling stages, the sucrose content in inferior grains was much higher than that in superior grains, and the poorer the grain filling, the higher the sucrose content was in inferior grains, indicating that the substrate concentration is not a limiting factor to their slow grain filling and poor grain plumpness for the inferior grains of two-line hybrid rice (Fig. 1, D-F). Changes of enzyme activities in grains SuSase activity was higher in superior grains than in inferior grains at early and mid grain filling Table 5. Characteristics of the vascular bundles of peduncle. No. of vascular Individual area (mm 2 ) Total area (mm 2 /culm) bundles Vascular bundle Phloem Xylem Vascular bundle Phloem Xylem Liangyoupeijiu b b c b b b b Yangliangyou a a a a a a a Shanyou b b b b b b b Within a column, data followed by the same lowercase letters indicated no significant difference at 0.05 level. Table 6. Vascular bundles, sink capacity and grain filling rate. Ratio of spikelets to total area of vascular bundles (No. of spikelets/mm 2 ) Ratio of spikelets to total area of phloem (No. of spikelets/mm 2 ) Ratio of grain weight to total area of phloem (g/mm 2 ) Ratio of sink to total area of phloem (g/mm 2 ) Transportation rate through phloem (g/mm 2 d) Liangyoupeijiu a a a a b Yangliangyou b b b b a Shanyou b b b b ab Within a column, data followed by the same lowercase letters indicated no significant difference at 0.05 level.

5 38 Rice Science, Vol. 13, No. 1, 2006 Fig. 1. Changes of starch (A-C) and sucrose (D-F) contents in rice grains. Vertical bars represent standard errors (n=3). Fig. 2. Changes in activities of sucrose synthase (SuSase, A-C) and acid invertase (AIV, D-F) in rice grains. Vertical bars represent standard errors (n=3). stages (Fig. 2, A-C). It reached the peak on DAF for superior grains, while on DAF for inferior ones. From 20 DAF on, it was significantly higher in inferior grains than in superior ones. Higher SuSase activity was corresponding with lower sucrose content in grains. AIV activity peaked on 8 DAF for superior grains, and on 12 DAF for inferior grains. From 4 DAF to 8 DAF, AIV in superior grains was higher than that in inferior grains, and from 12 DAF on, it was reversed. The difference in AIV activity was rather small within the same grain position among different combinations.

6 ZHAO Bu-hong, et al. Source-Sink and Grain-Filling Characteristics of Two-Line Hybrid Rice Yangliangyou 6 39 Changes in activities of AGPase, StSase and BE during grain filling period were as follows: Enzyme activities were low at initial filling, and increased firstly, then decreased sharply after reaching peak values (Fig. 3). Enzyme activities and the time reaching peak values varied with grain positions, combinations and enzyme types. At the early filling stage (4-12 DAF), activities and their peak values of three enzymes in superior grains were all higher than those in inferior grains. The time reaching peak value in superior grains was also earlier than that in inferior grains, however, at the mid or late grain filling stage (about DAF), enzyme activities in superior grains were higher than those in superior grains. For the combinations with high filling rate and good grain filling, enzyme activities in inferior grains were high at the early grain filling stage, and their peak values were also high. Among the three enzymes, AGPase and StSase tended to reach their peak values synchronously in the tested hybrid rice. The peaks of the superior and inferior grains appeared on 12 DAF and 20 DAF, respectively. The time for BE reaching its peak value was later than that for AGPase and StSase, and 16 DAF for superior grains and DAF for inferior grains (Fig. 3). Correlations of enzyme activities with grain filling rate Table 7 represented the coefficients of correlation of enzyme activities with the maximum grain filling rate (G max ), mean grain filling rate (G mean ), grainfilling degree (GFD), and gain weight (GW). G max, Liangyoupeijiu Liangyoupeijiu Liangyoupeijiu Adenosine diphosphoglucose pyrophosphorylase activity (U/ grain min) Yangliangyou Shanyou 63 Superior grains Inferior grains Starch synthase activity (U/ grain min) Yangliangyou Starch branching enzyme activity (U/ grain min) Yangliangyou Shanyou 63 Shanyou 63 Days after flowering (d) Days after flowering (d) Days after flowering (d) Fig. 3. Changes in adenosine diphosphoglucose pyrophosphorylase (AGPase, A-C), starch synthase (StSase, D-F) and starch branching enzyme (BE, G-I) activities in rice grains. Vertical bars represent standard errors (n=3).

7 40 Rice Science, Vol. 13, No. 1, 2006 Table 7. Correlations of the maximum and mean activities of SuSase, AIV, AGPase, StSase and BE in the grains with the maximum grain filling rate (G max), mean grain filling rate (G mean), grain-filling degree (GFD), and grain weight (GW). Item G max G mean GFD GW Maximum activity/content SuSase 0.921** 0.948** 0.931** 0.932** AIV AGPase 0.935** 0.941** 0.919** 0.952** StSase 0.923** 0.924** 0.923** 0.919** BE 0.977** 0.990** 0.954** 0.949** Mean activity/content SuSase 0.922** 0.935** 0.931** 0.924** AIV AGPase 0.929** 0.936** 0.925** 0.931** StSase 0.921** 0.918** 0.931** 0.924** BE 0.981** 0.978** 0.959** 0.941** ** Significant at 0.01 level (n=6). G mean, GFD and GW were very significantly and positively correlated with the peak activities of SuSase, AGPase, StSase and BE and their mean activities in active grain filling period (r = ** **, P < 0.01), but they were not significantly correlated with those of AIV activities (r = , P > 0.05). DISCUSSION Qi et al [9] reported that high matter accumulation before heading and high translocation rate after heading were the main reason for high yield in rice. Lin et al [10] found that relationship between dry matter accumulation at the heading and grain yield could be simulated by quadratic equation, and the difference in grain yield was due mainly to photosynthetic capacity from the heading stage to maturity stage. Yan et al [11] reported that heavy panicle hybrid rice had high photosynthetic productivity after heading, but translocation of assimilates was inefficient. Our results showed that the differences in dry matter accumulation before heading were rather small between two-line and three-line hybrid rice. However, the dry matter accumulation of two-line hybrid rice (LYPJ and YLY6) was significantly higher than that of three-line hybrid rice (SY63) from heading to maturity, indicating that two-line hybrid rice has superiority in matter production during the grain filling period. But the export percentage and transformation percentage of the matter in culms and sheaths of YLY6 and LYPJ were lower than those of SY63. We have also investigated the accumulation and translocation of assimilates on 28 two-line hybrid combinations and 10 three-line hybrid combinations, and the results were similar to this study (data not shown). We found that the dry matter accumulation was not significantly correlated with grain yield (r = ). Seed setting rate and grain filling degree were significantly or very significantly correlated with the export percentage and transformation percentage of the matter in culms and sheaths (r = * ** ). These results suggest that dry matter production is only the base of yield formation, and yield increase is not always synchronous with the increase in dry matter accumulation. On the base of higher biomass, the increase in the export percentage and transformation percentage of the matter in culms and sheaths is critical for improving grain filling of two-line hybrid rice, and it is also an important approach to transform photosynthetic potential to actual grain yield. Grain filling rate and grain-filling degree had obvious differences between superior and inferior grains in hybrid rice, especially in inter-subspecific hybrid rice and two-line hybrid rice [1, 15]. Differences in grain filling between superior and inferior grains were usually attributed to less substrate content [12-15]. However, our results demonstrated that, at the early

8 ZHAO Bu-hong, et al. Source-Sink and Grain-Filling Characteristics of Two-Line Hybrid Rice Yangliangyou 6 41 and mid grain filling stages, the sucrose content in inferior grains was greater than that in superior grains for the combinations with poor grain filling, indicating that the substrate concentration is not the principal factor for their slow grain filling and poor grain plumpness for the inferior grains of two-line hybrid rice. We speculated that low starch synthesis rate and slow starch accumulation are the main reasons for slow grain filling and poor grain plumpness in two-line hybrid rice. We discovered that the activities of SuSase, StSase, AGPase and BE were higher in superior grains than in inferior grains, and higher in the combination with good grain plumpness than that with poor grain plumpness. These results suggested that SuSase, AGPsase, StSase and BE in grains play an important role in grain filling, and the low grain filling rate and poor grain plumpness are closely associated with the low activities of the enzymes in inferior grains for some two-line hybrid rice combinations. It would be possible to breed varieties or combinations with good grain-filling by selecting parents with high activities of the enzyme in grains during the grain filling period, especially at the early filling stage. Both SuSase and AIV are involved in sucrose cleavage in sink tissue, their activities determine grain growth rate and have been regarded as the sink strength [16]. We found that SuSase activity was significantly higher than AIV activity at the early and mid grain filling stages. The grain filling rate, grain filling degree, and grain weight were positively and significantly correlated with the activity of SuSase, but not significantly correlated with AIV activity, indicating that SuSase plays an important role in sucrose-starch biosynthesis process. The mean and maximum activities of SuSase, AGPsase, StSase and BE during grain filling were positively and significantly or very significantly correlated with grain filling rate, grain filling degree, and grain weight. These results further proved the conclusion of these three enzymes were key enzymes for starch synthesis [5]. Among the three enzymes of AGPase, StSase and BE, AGPase is considered the rate-limiting enzyme in starch biosynthesis [17-18]. However, we found that the correlation coefficient of BE was the largest among all the correlations of activities of the three enzymes with grain filling rate, grain weight, and grain filling degree. From the process of starch synthesis, BE is not only involved in forming α -1,6-linked branches to synthesize amylopectin, but also in providingα-glucose with non-reducing end as acceptance, which is beneficial to catalyze reaction of AGPase and StSase, leading to an increase in starch synthesis rate [18]. Therefore, it may be concluded that BE plays a key role in grain filling, and its importance is possibly greater than AGPase s. There were many reports about relationship between the characteristics of peduncle tissues and big panicle [4, 19-21]. Our results showed that the greater the number of vascular bundles of peduncle, the greater the spikelet number per panicle, indicating that the increase in spikelet number is closely associated with the improvement of peduncle and culm tissues. Previous studies showed that poor grain filling was interrelated with the higher spikelet number load per unit area of vascular bundle of peduncle [20, 22]. We found that the greater the spikelet number, yield and total sink load per unit area of vascular bundle and phloem, the lower the seed setting rate and the poorer the grain filling, indicating that total sink load per unit area of phloem was an important index to reflect grain-filling situations. Based on the results from our study, we suggest that (1) great attention should be paid to breed variety with greater vascular bundle number and larger phloem area and to select a combination with strong tissues of peduncle, good grain filling, and great weight per panicle, so that to overcome the contradiction of large sink size and limited source. YLY6 is a successful case in breeding two-line hybrid rice, and (2) the spikelet number per panicle, grain yield and grain weight would be increased by raising strong seedlings, reducing unproductive tillers so as to form strong culm and promote the development of vascular bundles, and by improving fertilizer and water management at the late growth stage. Our results showed that grain-filling index comprehensively reflected source-sink characteristics during the grain filling period. It eliminates the interaction between the number of spikelets and grain weight to a certain extent, and also represents filledgrain rate and grain filling degree, so that it really

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