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Isolation and functional analysis of SrMYB1, a direct transcriptional repressor of SrUGT76G1 in Stevia rebaudiana

ZHANG Ting, ZHANG Yong-xia, SUN Yu-ming, XU Xiao-yang, WANG Yin-jie, CHONG Xin-ran, YANG Yong-heng and YUAN Hai-yan

2023, 22 (4): 1058-1067. DOI: 10.1016/j.jia.2023.03.001

Abstract （212） PDF in ScienceDirect

SrUGT76G1, the most well-studied diterpene glycosyltransferase in Stevia rebaudiana, is key to the biosynthesis of economically important steviol glycosides (SGs). However, the molecular regulatory mechanism of SrUGT76G1 has rarely been explored. In this study, we identified a MYB transcription factor, SrMYB1, using a yeast one-hybrid screening assay. SrMYB1 belongs to the typical R2R3-type MYB protein and is specifically localized in the nucleus with strong transactivation activity. The transcript of SrMYB1 is predominantly accumulated in flowers, but is also present at a lower level in leaves. Yeast one-hybrid and electrophoretic mobility shift assays verified that SrMYB1 binds directly to the MYB binding sites in the F4-3 fragment (+50–(–141)) of the SrUGT76G1 promoter. Furthermore, we found that SrMYB1 could significantly repress the expression of SrUGT76G1 in both epidermal cells of tobacco leaves and stevia callus. Taken together, our results demonstrate that SrMYB1 is an essential upstream regulator of SrUGT76G1 and provide novel insight into the regulatory network for the SGs metabolic pathway in S. rebaudiana.

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The nitrate-responsive transcription factor MdNLP7 regulates callus formation by modulating auxin response

LI Tong, FENG Zi-quan, ZHANG Ting-ting, YOU Chun-xiang, ZHOU Chao, WANG Xiao-Fei

2023, 22 (10): 3022-3033. DOI: 10.1016/j.jia.2023.08.007

Abstract （194） PDF in ScienceDirect

Under appropriate culture conditions, plant cells can regenerate new organs or even whole plants. De novo organ regeneration is an excellent biological system, which usually requires additional growth regulators, including auxin and cytokinin. Nitrate is an essential nutrient element for plant vegetative and reproductive development. It has been reported that nitrate is involved in auxin biosynthesis and transport throughout the growth and development of plants. In this study, we demonstrated that the ectopic expression of the MdNLP7 transcription factor in Arabidopsis could regulate the regeneration of root explants. MdNLP7 mainly participated in the regulation of callus formation, starting with pericycle cell division, and mainly affected auxin distribution and accumulation in the regulation process. Moreover, MdNLP7 upregulated the expression of genes related to auxin biosynthesis and transport in the callus formation stage. The results demonstrated that MdNLP7 may play a role in the nitrate-modulated regeneration of root explants. Moreover, the results revealed that nitrate–auxin crosstalk is required for de novo callus initiation and clarified the mechanisms of organogenesis.

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Potassium deficiency inhibits steviol glycosides synthesis by limiting leaf sugar metabolism in stevia (Stevia rebaudiana Bertoni) plants

SUN Yu-ming, HUANG Xiao-lei, ZHANG Ting, YANG Yong-heng, CHENG Xiao-fang, XU Xiao-yang, YUAN Hai-yan

2021, 20 (11): 2932-2943. DOI: 10.1016/S2095-3119(20)63472-4

Abstract （134） PDF in ScienceDirect

The steviol glycosides (SGs) in stevia (Stevia rebaudiana Bertoni) leaves are becoming increasingly valuable due to its high sweetness but low calorific value, which is driving the development of stevia commercial cultivation. Optimizing fertilization management can effectively increase SGs productivity, but knowledge on the relationship between potassium (K) fertilization and SGs production is still lacking. In this study, pot experiments were conducted in order to investigate the effect of K deficiency on SGs synthesis in stevia leaves, as well as the underlying mechanisms. Our results showed that when compared with standard K fertilization, K deficiency treatment has no significant effect on the biomass of stevia plant grown in a given soil with high K contents. However, K deficiency critically decreased leaf SGs contents as well as the expression of SGs synthesis-related genes. The contents of different sugar components decreased and the activities of sugar metabolism-related enzymes were inhibited under the K deficiency condition. Moreover, spraying sucrose on the leaves of stevia seedlings diminished the inhibitory effect caused by K deficiency. Our results also revealed the significant positive correlations between sucrose, glucose and SGs contents. Overall, our results suggest that K deficiency would suppress the synthesis of SGs in stevia leaves, and this effect may be mediated by the leaf sugar metabolism. Our findings provide new insights into the improvement of SGs production potential.

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Egg tanning improves the efficiency of CRISPR/Cas9-mediated mutant locust production by enhancing defense ability after microinjection

ZHANG Ting-ting, WEN Ting-mei, YUE Yang, YAN Qiang, DU Er-xia, FAN San-hong, Siegfried ROTH, LI Sheng, ZHANG Jian-zhen, ZHANG Xue-yao, ZHANG Min

2021, 20 (10): 2716-2726. DOI: 10.1016/S2095-3119(21)63736-X

Abstract （135） PDF in ScienceDirect

The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects. In the construction of CRISPR/Cas9-mediated dsLmRNase2^–/–mutant locusts, we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned. However, the heritable efficiency of the dsLmRNase2 deletion to the next generation G₁ progeny was similar between adults derived from the tanned or less tanned engineered eggs. Further, the similar effective mutant ratios in the normally developed eggs and G₀ adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9. Moreover, we found that the syncytial division period, which was longer than the time for tanning, conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs. We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection. Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure. In summary, tanned eggs possess stronger defense responses and higher efficiency of genome editing, providing an improved model for developing Cas9-mediated gene editing procedures in locusts.

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Identification of long-grain chromosome segment substitution line Z744 and QTL analysis for agronomic traits in rice

MA Fu-ying, DU Jie, WANG Da-chuan, WANG Hui, ZHAO Bing-bing, HE Guang-hua, YANG Zheng-lin, ZHANG Ting, WU Ren-hong, ZHAO Fang-ming

2020, 19 (5): 1163-1169. DOI: 10.1016/S2095-3119(19)62751-6

Abstract （118） PDF in ScienceDirect

Length of grain affects the appearance, quality, and yield of rice. A rice long-grain chromosome segment substitution line Z744, with Nipponbare as the recipient parent and Xihui 18 as the donor parent, was identified. Z744 contains a total of six substitution segments distributed on chromosomes (Chrs.) 1, 2, 6, 7, and 12, with an average substitution length of 2.72 Mb. The grain length, ratio of length to width, and 1 000-grain weight of Z744 were significantly higher than those in Nipponbare. The plant height, panicle number, and seed-set ratio in Z744 were significantly lower than those in Nipponbare, but they were still 78.7 cm, 13.5 per plant, and 86.49%, respectively. Furthermore, eight QTLs of different traits were identified in the secondary F₂ population, constructed by Nipponbare and Z744 hybridization. The grain weight of Z744 was controlled by two synergistic QTLs (qGWT1 and qGWT7) and two subtractive QTLs (qGWT2 and qGWT6), respectively. The increase in the grain weight of Z744 was caused mainly by the increase in grain length. Two QTLs were detected, qGL1 and qGL7-3, which accounted for 25.54 and 15.58% of phenotypic variation, respectively. A Chi-square test showed that the long-grain number and the short-grain number were in accordance with the 3:1 separation ratio, which indicates that the long grain is dominant over the short-grain and Z744 was controlled mainly by the principal effect qGL1. These results offered a good basis for further fine mapping of qGL1 and further dissection of other QTLs into single-segment substitution lines.

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Gene mapping and candidate gene analysis of aberrant-floral spikelet 1 (afs1) in rice (Oryza sativa L.)

ZHANG Ting, YOU Jing, YU Guo-ling, ZHANG Yi, CHEN Huan, LI Yi-dan, YE Li, YAO Wan-yue, TU Yu-jie, LING Ying-hua, HE Guang-hua, LI Yun-feng

2020, 19 (4): 921-930. DOI: 10.1016/S2095-3119(19)62847-9

Abstract （136） PDF in ScienceDirect

The spikelet is a unique inflorescence structure in grasses. However, the molecular mechanism that regulates its development remains unclear, and we therefore characterize a spikelet mutant of rice (Oryza sativa L.), aberrant-floral spikelet 1 (afs1), which was derived from treatment of Xinong 1B with ethyl methanesulfonate. In the afs1 mutant, the spikelet developed an additional lemma-like organ alongside the other normally developed floral organs, and the paleae were degenerated to differing degrees with or without normally developed inner floral organs. Genetic analysis revealed that the afs1 phenotype was controlled by a single recessive gene. The AFS1 gene was mapped between the insertion/deletion (InDel) marker Indel19 and the simple sequence repeat marker RM16893, with a physical distance of 128.5 kb on chromosome 4. Using sequence analysis, we identified the deletion of a 5-bp fragment and a transversion from G to A within LOC_Os04g32510/ LAX2, which caused early termination of translation in the afs1 mutant. These findings suggest that AFS1 may be a new allele of LAX2, and is involved in the development of floral organs by regulating the expression of genes related to their development. The above results provide a new view on the function of LAX2, which may also regulate the development of spikelets.

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Screening of antagonistic Trichoderma strains and their application for controlling stalk rot in maize

LU Zhi-xiang, TU Guang-ping, ZHANG Ting, LI Ya-qian, WANG Xin-hua, Zhang Quan-guo, SONG Wei, CHEN Jie

2020, 19 (1): 145-152. DOI: 10.1016/S2095-3119(19)62734-6

Abstract （181） PDF in ScienceDirect

Maize is one of the major crops in China, but maize stalk rot occurs nationwide and has become one of the major challenges in maize production in China. In order to find an environment-friendly and feasible technology to control this disease, a Trichoderma-based biocontrol agent was selected. Forty-eight strains with various inhibition activities to Fusarium graminearum, and Fusarium verticillioides were tested. A group of Trichoderma strains (DLY31, SG3403, DLY1303 and GDFS1009) were found to provide an inhibition rate to pathogen growth in vitro of over 70%. These strains also prevented pathogen infection over 65% and promoted the maize seedling growth for the main root in vivo by over 50%. Due to its advantage in antifungal activity against pathogens and promotion activity to maize, Trichoderma asperellum GDSF1009 was selected as the most promising strain of the biocontrol agent in the Trichoderma spectrum. Pot experiments showed that the Trichoderma agent at 2–3 g/pot could achieve the best control of seedling stalk rot and promotion of maize seedling growth. In the field experiments, 8–10 g/hole was able to achieve over 65% control to stalk rot, and yield increased by 2–11%. In the case of natural morbidity, the control efficiency ranged from 27.23 to 48.84%, and the rate of yield increase reached 11.70%, with a dosage of Trichoderma granules at 75 kg ha–1. Based on these results, we concluded that the Trichoderma agent is a promising biocontrol approach to stalk rot in maize.

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Identification and validation of root-specific promoters in rice

HUANG Li-yu, ZHANG Fan, QIN Qiao, WANG Wen-sheng, ZHANG Ting, FU Bin-ying

2015, 14 (1): 1-10. DOI: 10.1016/S2095-3119(14)60763-2

Abstract （2238） PDF in ScienceDirect

Novel promoters that confer root-specific expression would be useful for engineering resistance against problems of nutrient and water absorption by roots. In this study, the reverse transcriptase polymerase chain reaction was used to identify seven genes with root-specific expression in rice. The isolation and characterization of upstream promoter regions of five selected genes rice root-specific promoter (rRSP) 1 to 5 (rRSP1-rRSP5) and A2P (the promoter of OsAct2) revealed that rRSP1, rRSP3, and rRSP5 are particularly important with respect to root-specific activities. Furthermore, rRSP1, rRSP3, and rRSP5 were observed to make different contributions to root activities in various species. These three promoters could be used for root-specific enhancement of target gene(s).

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