Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (24): 4781-4792.doi: 10.3864/j.issn.0578-1752.2022.24.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Phenotypic Analysis and Gene Cloning of Rice Panicle Apical Abortion Mutant paa21

HE Lei1(),LU Kai1,ZHAO ChunFang1,YAO Shu1,ZHOU LiHui1,ZHAO Ling1,CHEN Tao1,ZHU Zhen1,ZHAO QingYong1,LIANG WenHua1,WANG CaiLin1,ZHU Li2(),ZHANG YaDong1()   

  1. 1Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/East China Branch of National Center of Technology Innovation for Saline-Alkali Tolerant Rice/Jiangsu High Quality Rice R&D Center/Nanjing Branch of China National Center for Rice Improvement/Key laboratory of Jiangsu Province for Agrobiology, Nanjing 210014
    2China National Rice Research Institute/State Key Laboratory of Rice Biology, Hangzhou 310006
  • Received:2022-09-27 Accepted:2022-10-24 Online:2022-12-16 Published:2023-01-04
  • Contact: Li ZHU,YaDong ZHANG E-mail:helei@jaas.ac.cn;zhuli05@caas.cn;zhangyd@jaas.ac.cn

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Abstract:

【Objective】Rice panicle apical abortion affects yield. Identification and cloning of genes related to rice panicle apical abortion can enrich the molecular mechanism of rice panicle development regulation, and provide theoretical basis and genetic resources for rice high-yield molecular design breeding. 【Method】Here, a stably inherited panicle apical abortion 21 (paa21) mutant was screened from EMS mutant library of the japonica rice variety "Wuyunjing 30". Agronomic traits, such as ratio of degraded primary branches, degraded apical spikelets, grains per panicle, plant height, panicle length, and grain yield per plant, were statistically analyzed. Trypan blue and Evans blue staining were used to detect whether programmed cell death occurred in the apical spikelets. H2O2 content in young panicles at different development stages and different panicle parts of WT and paa21 was determined. Genetic analysis was carried out by reciprocal cross of paa21 with indica rice II-32B and 9311 respectively. The F2 population constructed by crossing paa21 with indica rice II-32B was used for gene mapping and cloning. The three-dimensional structure of wild-type and paa21 proteins were predicted using SWISS-MODEL website. The expression levels of ROS response marker genes, programmed cell death related genes and catalase related genes were analyzed by RT-qPCR. 【Result】paa21 produced panicle apical abortion phenotype and the degenerated spikelets were mainly located on the primary branches at the apical panicle. The plant height, grain number per panicle, panicle length and grain yield per plant of paa21 were lower than those of WT. After observing the young panicles at different development stages, we found that the paa21 mutant had a panicle apical abortion phenotype when panicle developed to 12 cm. Trypan blue and Evans blue staining results showed that the apical spikelets of the paa21 mutant had programmed cell death. Stronger DAB staining was observed in the degenerated apical spikelets of paa21 than WT. The results of H2O2 content determination showed that higher level of ROS was accumulated in panicle of paa21 compared with WT. Genetic analysis suggested that paa21 mutant phenotype is controlled by a pair of recessive nuclear genes. The results of map-based cloning showed that a C to T mutation occurred in the second exon of Os02g0673100 in paa21, resulting in the mutation of alanine to valine. This gene encodes an aluminum activated malate transporter, ALMT7. The mutation site was located at the fourth transmembrane helix. SWISS-MODEL prediction results showed that the mutation site did not significantly affect the three-dimensional structure of the mutant protein. The expression level of ROS response marker genes Os01g0826400, Os05g0474800 and Os02g0181300 in paa21 was significantly higher than that in WT when the young spike developed to 10 cm. Compared with WT, the expression level of programmed cell death related genes VPE2 and VPE3 increased significantly in paa21. The expression level of CATA, CATB and CATC which encode catalase in 10 cm young panicle of paa21 was significantly higher than that of WT. The activity of CAT in paa21 10 cm young spikelet was significantly lower than that of WT. 【Conclusion】paa21 accumulate excess ROS in the apical spikelet at late stage of panicle development, resulting in programmed cell death, which eventually leads to the degeneration of the apical spikelet. These results lay a good foundation for further enriching the genetic regulatory network of panicle development.

Key words: rice, panicle apical abortion, reactive oxygen species, programmed cell death

Fig. 1

Phenotypic characterization of the paa21 mutant A: Morphology of WT (left) and paa21 (right), Bars=10 cm; B: Panicle of WT (left) and paa21 (right) at heading stage, Bars=2 cm; C: Panicle of WT (left) and paa21 (right) at mature stage, Bars=2 cm; D: Primary branches from one panicle, 1-16 indicate primary branches from the top to the bottom of the panicle in paa21; E: Statistics for normal spikelets and degenerated spikelets correspond to Fig. D; F: Ratio of degenerated primary branches; G: Ratio of degenerated grains per panicle of WT and paa21; H: Quantification of plant height; I: Quantification of grain number per panicle; J: Quantification of panicle length; K: Quantification of grain yield per plant. Error bars mean standard error; ** P≤0.01 by the Student’s t-test. The same as below"

Fig. 2

Developing panicles of WT and paa21 A-F: Developing panicles of WT (left) and paa21 (right). 1 cm (A), 2 cm (B), 5 cm (C), 7 cm (D), 12 cm (E), 17 cm (F). Bars=1 cm"

Fig. 3

Trypan blue and Evans blue staining of apical spikelets in WT and paa21 A: Trypan blue staining; B: Evans blue staining"

Fig. 4

Detection of H2O2 accumulation A: DAB staining of apical spikelets in WT and paa21 panicle; B: H2O2 content in apical spikelets of WT and paa21 at different stages of panicle development, PL; C: H2O2 content in spikelets of different parts of rice panicle, Different letters indicate significantly different values determined using analysis of variance followed by an LSD test for the comparison of means (P<0.05), ap: Apical part, mp: Middle part, bp: Bottom part"

Table 1

Genetic analysis of the paa21"

组合
Crossing combination		 F1表型
Phonotype of F1 plants		 F2单株数 Number of F2 plants χ2（3﹕1=3.84）
野生型 Wild type 突变型 Mutant type
paa21×II-32B 野生型Wild type 396 129 0.0514
II-32B×paa21 野生型Wild type 516 182 0.4298
paa21×9311 野生型Wild type 253 81 0.0998
9311×paa21 野生型Wild type 474 164 0.1693

Fig. 5

Map-base cloning of PAA21"

Fig. 6

Amino acid sequence alignment of WT and paa21(A) and predicted 3D structure of WT and paa21 proteins (B, C) M1-M6 represent the transmembrane helices of PAA21 protein, and the red box indicates the mutation site"

Fig. 7

Expression levels of ROS, PCD-related genes, and CAT activity"

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