Abscisic acid and the key enzymes and genes in sucrose-to-starch conversion in rice spikelets in response to soil-drying during grain filling

Planta

Zhiqin Wanga · Yunji Xua · Tingting Chena · Hao Zhanga · Jianchang Yanga · Jianhua Zhangb

a Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China

b School of Life Sciences and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, China

Correspondence to: J. Yang, Tel: +86 514 87324276, Fax: +86 514 87979317, E-mail: /J. Zhang, Tel: +852 3943 6288, Fax: +852 2603 6382, E-mail:

Electronic Supplementary Material Description:

This document contains the primers sequence of the genes determined in the study (Table S1), the data on grain yield and yield components (Table S2), correlation coefficients of the grain filling rate with the activities and gene expression levels of the enzymes involved in sucrose-to-starch conversion (Table S3), the data on effects of applied ABA and fluridone on the activities and gene expression levels of the enzymes (Table S4), the data on soluble carbohydrate content in the flag leaf (Fig. S1) and chlorophyll content in the flag leaf of rice (Fig. S2).

Table S1 The forward (F) and reverse (R) primers sequence of genes in sucrose-to-starch conversion and the genes encoding 9-cis-epoxycarotenoid dioxygenase in rice grains

Gene name / Accession number / Primer pairs / Product size (bP)
CELL-WALL INVERTASE 1 (OsCIN1) / AB073749 / F:5’- ATTGACCGTTCGGTGGTTGA-3’
R:5’- GGCCGGTATATCGCAGATGG-3’ / 270
CELL-WALL INVERTASE 4 (OsCIN4) / AB154521 / F:5’-CAGTAGCACGGTGAAGGTGT-3’
R:5’-CCAATTGCGAACCATTTCTTAAC-3’ / 320
VACUOLAR INVERTASE 2 (INV2) / AF276703 / F:5’- CCATCTACGAGTCGGCCAAA-3’
R:5’- CGCATGCGCACACACAATAA-3’ / 307
SUCROSE SYNTHASE 2 (SuS2) / AK072074 / F:5’-AGAGGCTCACTGCTTTCCAC-3’
R:5’-CATCTCCCCGGTACGAACAC-3’ / 350
SUCROSE SYNTHASE 3 (SuS3) / AK100306 / F:5’- GTTGAGGAGCTGAGTGTCCC-3’
R:5’- TGTGCGCACGAAGAAAGTTG-3’ / 238
SUCROSE SYNTHASE 4 (SuS4) / AK102158 / F:5’- TCGAAACTCCCAGCTGACAC-3’
R:5’- CCAACCCTTCTCCAATCCCC-3’ / 75
ADP-GLUCOSE PYROPHOSPHORYLASE SMALL SUBUNIT 1 (AGPS1) / AY028315 / F:5’- GAGACTGGTGGCATTCCCAT-3’
R:5’- AGGCCCAACAGTGGTGATTG-3 / 237
ADP-GLUCOSE PYROPHOSPHORYLASE SMALL SUBUNIT 2b (AGPS2b) / AK103906 / F:5’- ACTCACCACTGGCCTCACTT-3’
R:5’-TGCACCACCTCCAAGAATGA-3’ / 387
ADP-GLUCOSE PYROPHOSPHORYLASE LARGE SUBUNIT 1 (AGPL1) / AK100910 / F:5’- GCATGCGTGAGCCCAATAAG-3’
R:5’- TGGATCCGCAAAGTTCCTCC-3’ / 216
ADP-GLUCOSE PYROPHOSPHORYLASE LARGE SUBUNIT 2 (AGPL2) / AK071497 / F:5’- ACCAGAGGGTGCTGATTTGG-3’
R:5’- CCAAAGTCTTGCAGGTGTGC-3’ / 171
SOLUBLE STARCH SYNTHASE IIa (SSSIIa) / AK101978 / F:5’- ACCGCAAGTACAAGGAGAGC-3’
R:5’- ACCATGCCTCGCCATAAACA-3’ / 333
SOLUBLE STARCH SYNTHASE IIb (SSSIIb) / AK066446 / F:5’- GACGTGCAGGTGGTGATGG-3’
R:5’- CCCCTCCAGCTCTCCTTGTA-3’ / 382
SOLUBLE STARCH SYNTHASE IIc (SSSIIc) / AK072339 / F:5’- ACCAGAGCAATGTGAGGAGC-3’
R:5’- TGTCGGTGGAAGAGAAGTGC-3’ / 221
GRANULE-BOUND STARCH SYNTHASE I (GBSSI) / AK070431 / F:5’- ACCAGTACAAGGACGCTTGG-3’
R:5’- GCAAAGAAGGCTGAAACGCA-3’ / 227
GRANULE-BOUND STARCH SYNTHASE II (GBSSII) / AK102058 / F:5’-TTATGCCTCGCTGCTTTGGA-3’
R:5’-TGTGCCAGTCATTTGCAACG-3’ / 109
STARCH BRANCHING ENZYME I (SBEI) / AK065121 / F:5’-GCCCCTTCCCTGATTCTTCT-3’
R:5’-AAACCATCCTCGCTACCAGC-3’ / 216
STARCH BRANCHING ENZYME IIa (SBEIIa) / AB023498 / F:5’-GGAATGAGTAGCCCGGAACC-3’
R:5’- AAGAGTGCTCCTGGATTGCC-3’ / 121
STARCH BRANCHING ENZYME IIb (SBEIIb) / D16201 / F:5’-GGCGTGAAGAGAGTGGTTGA -3’
R:5’-CCATCTCCTGTTGGTGGGAC-3’ / 71
9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (NCED1) / AK064824 / F:5’-TGACGTACTTCCGGTTCGAC-3’
R:5’-CTCGATGGAGAGGACTTCGG-3’ / 353
9-CIS-EPOXYCAROTENOID DIOXYGENASE 5 (NCED5) / AK107649 / F:5’-AAAGGCTTTACCAGAGGGCG-3’
R:5’-GAGCTCTCCTTGGCTGTCTG-3’ / 252
ACTIN / AY212324.1 / F:5’-GTGATGGTTGGTATGGGGCA-3’
R:5’-CTCAGTCAGCAACACAGGGT-3’ / 195

Table S2 Grain yield and yield components of rice under various soil-drying treatments

Treatment / Number of spikelets
(×104 m-2) / Filled grains
(%) / Grain weight
(mg) / Grain yield
(g m-2)
WW / 3.88±0.21a / 88.3±1.24b / 27.2±0.45b / 932±24.5b
MD / 3.87±0.19a / 93.9±1.67a / 28.6±0.51a / 1039±41.3a
SD / 3.85±0.17a / 82.5±1.48c / 24.8±0.46c / 787±20.7c

Different letters indicate statistical difference at P = 0.05 level within the column

Table S3 Correlations of the grain filling rate with the activities and gene expression levels of the enzymes involved in sucrose-to-starch conversion in rice spikelets during the active grain filling period

Data are from Figs 2b, 3 a-c, 4a-f, 5 a-d, 6 a-d, and 7 a-h. WW, MD and SD represent well-watered, moderate soil-drying and severe soil-drying treatments, respectively, during grain filling

Correlations with / Grain filling rate
WW / MD / SD / All treatments
Enzymatic activity
Sucrose synthase / 0.79** / 0.83** / 0.77** / 0.78**
Soluble acid invertase / 0.24 / 0.22 / 0.16 / 0.14
Insoluble acid invertase / 0.25 / 0.37 / 0.26 / 0.29
ADP-glucose pyrophosphorylase / 0.77** / 0.81** / 0.69* / 0.75**
Soluble starch synthase / 0.66* / 0.79** / 0.68* / 0.67**
Granule-bound starch synthase / 0.71** / 0.78** / 0.67* / 0.70**
Starch branching enzyme / 0.73** / 0.75** / 0.74** / 0.74**
Gene expression level
SuS2 / 0.79** / 0.82** / 0.75** / 0.79**
SuS3 / 0.86** / 0.91** / 0.72** / 0.81**
SuS4 / 0.71** / 0.76** / 0.72** / 0.70**
OsCIN1 / 0.27 / 0.33 / 0.23 / 0.28
OsCIN4 / 0.26 / 0.31 / 0.20 / 0.26
INV2 / 0.03 / 0.08 / 0.01 / 0.02
AGPS1 / 0.38 / 0.32 / 0.28 / 0.31
AGPS2b / 0.68* / 0.72** / 0.67* / 0.66**
AGPL1 / 0.77** / 0.80** / 0.67* / 0.73**
AGPL2 / 0.81** / 0.89** / 0.70* / 0.79**
SSSIIa / 0.25 / 0.49 / 0.36 / 0.31
SSSIIb / 0.86** / 0.90** / 0.76** / 0.83**
SSSIIc / 0.79** / 0.84** / 0.71** / 0.76**
GBSSI / 0.81** / 0.87** / 0.69* / 0.78**
GBSSII / 0.74** / 0.77** / 0.67* / 0.72**
SBEI / 0.81** / 0.86** / 0.70* / 0.78**
SBEIIa / 0.48 / 0.22 / 0.47 / 0.30
SBEIIb / 0.76** / 0.85** / 0.65* / 0.75**

*,** Correlation significance at P = 0.05 and P = 0.01 levels, respectively (n = 12 for the WW, MD or SD treatment, and n = 36 for all the treatments)

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Table S4 Effects of applied abscisic acid (ABA) and fluridone on the activities of some enzymes involved in sucrose-to-starch conversion and the expression levels of the genes encoding the enzymes in rice spikelets

Enzyme activity/ gene expression / 12 d after heading / 18 d after heading
CK / T1 / T2 / T3 / T4 / CK / T1 / T2 / T3 / T4
Superior spikelets
Soluble AI / 16.3±0.37a / 17.1±0.82a / 15.8±0.73a / 12.5±0.60b / 16.7±0.71a / 6.31±0.29a / 6.24±0.37a / 6.14±0..46a / 3.48±0.25b / 6.28±0.53a
Insoluble AI / 12.7±0.58a / 13.2±0.96a / 12.4±0.71a / 8.53±0.45b / 11.8±0.82a / 7.42±0.35a / 7.26±0.47a / 7.72±0..56a / 4.67±0.34b / 7.39±0.56a
OsCIN1 / 155±11.6a / 163±12.7a / 147±12.5a / 124±10.2b / 159±14.5a / 57.5±3.23a / 60.8±4.29a / 55.6±4.17a / 36.2±2.72b / 58.3±3.33a
OsCIN4 / 117±8.65a / 121±7.69a / 115±10.2a / 83.4±7.21b / 114±6.57a / 65.8±4.16a / 68.1±4.76a / 62.5±3.53a / 45.8±3.29b / 63.5±4.82a
INV2 / 37.8±2.27a / 36.4±2.32a / 35.9±2.37a / 24.5±1.63b / 38.1±2.48a / 32.4±1.79a / 33.2±2.08a / 31.3±2.24a / 21.9±1.52b / 32.9±2.24a
AGPS1 / 115±10.4a / 119±10.7a / 107±9.41a / 87.6±5.45b / 120±11.3a / 85.7±4.50a / 87.4±4.29a / 83.2±4.76a / 60.5±3.45b / 83.5±4.88a
AGPS2b / 124±10.7a / 127±11.5a / 120±10.2a / 91.2±7.21b / 122±10.5a / 47.5±2.31a / 49.4±2.73a / 44.7±3.18a / 30.5±3.17b / 44.3±3.19a
AGPL1 / 107±9.31a / 116±10.3a / 84.5±5.52b / 63.4±5.30c / 112±10.6a / 43.3±2.52a / 44.9±3.14a / 31.1±2.47b / 22.5±1.93c / 45.7±3.72a
SuS2 / 152±12.4a / 157±11.8a / 127±10.6b / 105±9.86c / 150±10.3a / 37.6±2.45a / 40.5±2.92a / 28.4±1.75b / 21.7±1.34c / 38.6±1.97a
SuS4 / 134±10.2a / 139±10.4a / 105±8.50b / 78.2±5.47c / 140±11.6a / 40.4±2.64a / 42.7±2.85a / 25.8±1.99b / 16.3±1.05c / 39.9±1.78a
SSSIIa / 105±6.46a / 107±7.21a / 98.6±6.19a / 81.7±4.54b / 102±7.55a / 53.2±3.70a / 54.8±3.01a / 51.5±3.32a / 36.9±226b / 50.8±3.45a
SSSIIc / 74.6±4.52a / 77.5±5.37a / 72.4±4.38a / 62.3±3.35b / 75.3±5.24a / 34.7±2.57a / 36.2±2.31a / 33.9±2.13a / 21.4±1.95b / 33.4±2.83a
GBSSII / 106±11.8a / 112±10.3a / 84.5±5.21b / 63.6±4.49c / 108±8.73a / 34.5±2.39a / 36.2±2.70a / 24.7±2.85b / 17.2±1.67c / 33.5±2.36a
SBEIIa / 67.8±3.35a / 69.6±3.26a / 65.5±3.45a / 51.8±2.92b / 64.9±3.05a / 48.2±2.45a / 49.6±2.38a / 47.4±2.34a / 37.7±2.08b / 47.4±2.46a
SBEIIb / 166±12.7a / 175±14.4a / 135±11.6b / 105±8.48c / 161±12.5a / 83.4±5.32a / 87.4±5.27a / 69.5±4.13b / 50.3±3.54c / 85.6±5.62a
Inferior spikelets
Soluble AI / 14.9±0.45a / 15.2±0.76a / 14.2±0.61a / 11.8±0.52b / 15.0±0.64a / 7.27±0.35a / 7.38±0.43a / 6.97±0..51a / 3.75±0.29b / 7.12±0.49a
Insoluble AI / 13.2±0.63a / 13.9±0.72a / 12.8±0.66a / 9.31±0.48b / 12.9±0.55a / 8.10±0.42a / 8.29±0.51a / 7.87±0.48a / 4.79±0.36b / 7.95±0.47a
OsCIN1 / 161±12.4a / 169±13.8a / 154±11.8a / 127±11.5b / 156±12.7a / 65.2±3.56a / 68.6±3.94a / 61.5±4.39a / 46.3±3.67b / 62.8±4.65a
OsCIN4 / 122±9.28a / 127±8.43a / 118±9.50a / 87.5±6.79b / 125±8.63a / 68.3±4.67a / 70.5±4.82a / 64.9±3.85a / 50.2±3.11b / 66.7±3.54a
INV2 / 76.5±4.42a / 77.3±4.31a / 75.8±4.15a / 57.2±2.46b / 75.8±4.20a / 53.6±2.45a / 55.3±2.93a / 51.3±3.47a / 36.5±2.02b / 54.3±272a
AGPS1 / 67.8±3.33a / 69.0±4.14a / 66.4±4.77a / 51.6±2.53b / 65.9±3.75a / 41.4±2.62a / 42.6±2.49a / 39.5±2.70a / 28.7±2.13b / 43.2±2.64a
AGPS2 / 76.4±3.45b / 85.2±4.03a / 67.6±2.94c / 54.2±2.86d / 74.3±3.20b / 95.0±5.14b / 112±7.65a / 82.4±4.41c / 66.9±3.15d / 93.8±4.51b
AGPL1 / 34.6±2.55b / 45.7±2.62a / 25.3±1.74c / 18.2±1.26d / 36.3±1.81b / 97.5±5.73b / 114±7.76a / 82.6±4.91c / 62.4±3.42d / 119±9.73b
SuS2 / 43.7±2.82b / 52.8±3.17a / 33.6±2.65c / 24.8±1.84d / 42.5±2.71b / 87.8±5.11b / 104±8.49a / 74.2±5.26c / 60.7±3.75d / 85.4±4.18b
SuS4 / 48.5±2.91b / 57.6±2.99a / 36.7±2.83c / 25.6±1.71d / 46.0±2.45b / 90.4±6.06b / 111±7.58a / 72.5±4.72c / 57.8±3.29d / 87.6±5.45b
SSSIIa / 84.3±5.05a / 86.7±5.39a / 82.5±4.89a / 70.9±3.60b / 83.4±4.93a / 59.6±3.13a / 60.5±3.58a / 56.8±4.17a / 39.5±285b / 55.3±3.52a
SSSIIc / 41.4±2.87b / 54.1±3.34a / 32.3±2.45c / 22.7±1.53d / 43.3±2.86b / 82.5±5.24b / 97.6±5.65a / 70.3±3.42c / 52.4±2.73d / 95.5±5.71b
GBSSII / 39.8±2.35b / 48.9±2.75a / 32.6±2.04c / 24.5±1.97d / 40.8±2.54b / 74.4±4.19b / 88.7±5.43a / 63.6±3.91c / 48.7±2.74d / 73.9±4.86b
SBEIIa / 56.5±2.81a / 57.8±3.13a / 54.7±3.22a / 44.9±2.82b / 55.5±2.95a / 46.6±2.50a / 47.5±2.14a / 45.1±2.28a / 35.2±2.35b / 45.8±2.57a
SBEIIb / 77.9±4.46b / 92.3±6.84a / 68.5±3.06c / 52.6±2.42d / 78.5±3.18b / 83.7±4.33b / 97.6±6.57a / 72.5±4.02c / 57.9±2.87d / 82.7±4.49b

Different letters indicate statistical significance at P = 0.05 level (n = 8) within the same row and the same determination date

Fig. S1 Changes in soluble carbohydrate content in the flag leaf of rice. WW, MD and SD represent well-watered, moderate soil-drying and severe soil-drying treatments, respectively, during grain filling. Vertical bars represent LSD0.05 (DF=15)

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Fig. S2 Changes in chlorophyll content in the flag leaf of rice. WW, MD and SD represent well-watered, moderate soil-drying and severe soil-drying treatments, respectively, during grain filling. Vertical bars represent LSD0.05 (DF=15)

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