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. 2022 Dec 20:13:1042379.
doi: 10.3389/fpls.2022.1042379. eCollection 2022.

A promoter toolbox for tissue-specific expression supporting translational research in cassava (Manihot esculenta)

Wolfgang Zierer  1 , Ravi Bodampalli Anjanappa  2 , Christian Erwin Lamm  1 , Shu-Heng Chang  3 , Wilhelm Gruissem  2   3 , Uwe Sonnewald  1
Affiliations

A promoter toolbox for tissue-specific expression supporting translational research in cassava (Manihot esculenta)

Wolfgang Zierer et al. Front Plant Sci. .

Erratum in

Abstract

There is an urgent need to stimulate agricultural output in many tropical and subtropical countries of the world to combat hunger and malnutrition. The starchy crop cassava (Manihot esculenta), growing even under sub-optimal conditions, is a key staple food in these regions, providing millions of people with food. Cassava biotechnology is an important technique benefiting agricultural progress, but successful implementation of many biotechnological concepts depends on the availability of the right spatiotemporal expression tools. Yet, well-characterized cassava promoters are scarce in the public domain. In this study, we investigate the promoter activity and tissue specificity of 24 different promoter elements in stably transformed cassava plants. We show that many of the investigated promoters, especially from other species, have surprisingly low activity and/or tissue specificity, but feature several promoter sequences that can drive tissue-specific expression in either autotrophic-, transport- or storage tissues. We especially highlight pAtCAB1, pMePsbR, and pSlRBCS2 as strong and specific source promoters, pAtSUC2, pMeSWEET1-like, and pMeSUS1 as valuable tools for phloem and phloem parenchyma expression, and pStB33, pMeGPT, pStGBSS1, as well as pStPatatin Class I, as strong and specific promoters for heterotrophic storage tissues. We hope that the provided information and sequences prove valuable to the cassava community by contributing to the successful implementation of biotechnological concepts aimed at the improvement of cassava nutritional value and productivity.

Keywords: biotechnology; cassava; parenchyma; phloem; promoter; storage root; tissue; xylem.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Summary of the approximate promoter activity of ten different promoters in source leaves, stem, and storage root. (A) Composition of the seven multigene constructs analyzed for gene expression of the individual target genes (target genes not shown). Orange indicates promoter choice for desired expression in heterotrophic tissues, green indicates promoter choice for desired expression in autotrophic tissues. (B) The relative gene expression (normalized to MeGAPDH) of different transcripts was determined and the data was used to infer the approximate activity of the promoter element controlling its expression. Field-grown cassava plants were used to sample fully exposed source leaves (in the afternoon), stem pieces at the lower end of the first branching point, and storage root material from the two thickest storage roots per plant.
Figure 2
Figure 2
Representative GUS staining pattern of at least three events from pAtCAB1, pStLS1, pAtRBCS3B, pMeGBSS1, pStSSS3, and pStSTP1 promoter-reporter plants. pAtCAB1::GUS = A1 ) Source leaf (Inlay = Close-up), B1 ) Sink leaf, C1 ) Emerging leaves, D1 ) Petiole crosssection, E1 ) Upper stem cross-section, F1 ) Lower stem cross-section, G1 ) Storage root cross-section, H1 ) Fibrous roots, I1 ) GUS expression levels of four pAtCAB1::GUS lines relative to three pCaMV35S::GUS lines in %. pStLS1::GUS = A2 ) Source leaf, B2 ) Sink leaf, C2 ) Petiole cross-section, D2 ) Upper stem cross-section, E2 ) Storage root cross-section, F2 ) Fibrous roots. pAtRBCS3B::GUS = A3 ) Source leaf, B3 ) Sink leaf, C3 ) Petiole cross-section, D3 ) Upper stem cross-section, E3 ) Storage root cross-section, F3 ) Fibrous roots. pMeGBSS1::GUS = A4 ) Source leaf, B4 ) Sink leaf, C4 ) Petiole cross-section, D4 ) Upper stem cross-section, E4 ) Storage root cross-section, F4 ) Fibrous roots. pStSSS3::GUS = A5 ) Source leaf, B5 ) Sink leaf, C5 ) Petiole cross-section, D5 ) Upper stem cross-section, E5 ) Storage root cross-section, F5 ) Fibrous roots. pStSTP1::GUS = A6 ) Source leaf, B6 ) Sink leaf, C6 ) Petiole cross-section, D6 ) Upper stem cross-section, E6 ) Storage root cross-section, F6 ) Fibrous roots. Plants were either grown on the field at NCHU experimental station Taichung, Taiwan or in a greenhouse in Erlangen, Germany. Tissues from approximately 3-month-old cassava plants were used.
Figure 3
Figure 3
Representative GUS staining pattern of three pMePsbr::GUS promoter-reporter lines. (A) Source leaf, (B) Sink leaf, (C) Emerging leaves, (D) Petiole cross-section, (E) Upper stem cross-section, (F) Lower stem cross-section, (G) Storage root cross-section, (H) Fibrous roots, (I) GUS expression levels of three pPsbR::GUS lines relative to three pCaMV35S::GUS lines in %. Bars represent mean values with standard deviation (n=4).
Figure 4
Figure 4
Representative GUS staining pattern of four pAtSUC2::GUS promoter-reporter lines. (A) Source leaf (Inlay = Close-up), (B) Sink leaf (Inlay = Close-up), C Emerging leaves, (D) Petiole cross-section, (E) Upper stem cross-section, (F) Lower stem cross-section, (G) Storage root cross-section, (H) Fibrous roots (Inlay = Root tip).
Figure 5
Figure 5
Representative GUS staining pattern of four pCmGolS1 promoter-reporter lines. (A) Source leaf (Inlay = Close-up), (B) Sink leaf (Inlay = Close-up), (C) Emerging leaves, (D) Petiole cross-section, (E) Upper stem cross-section, (F) Lower stem cross-section, (G) Storage root cross-section (Inlay = Close-up), (H) Fibrous roots (Inlay = Root tip).
Figure 6
Figure 6
Representative GUS staining pattern of four pCoYMV promoter-reporter lines. (A) Source leaf (Inlay = Close-up), (B) Sink leaf (Inlay = Close-up), (C) Emerging leaves, (D) Petiole cross-section (Inlay = Close-up), (E) Upper stem cross-section (Inlay = Close-up), (F) Lower stem cross-section (Inlay = Close-up), (G) Storage root cross-section (Inlay = Close-up), (H) Fibrous roots (Inlay = Root tip).
Figure 7
Figure 7
Representative GUS staining pattern of at least four pMeSWEET1-like promoter-reporter lines. (A) Source leaf, (B) Sink leaf, (C) Emerging leaves, (D) Petiole cross-section, (E) Upper stem cross-section (Inlay = Close-up), (F) Lower stem cross-section (Inlay = Close-up), (G) Storage root cross-section (Inlay = Close-up), (H) Fibrous roots (Inlay = Developing side root).
Figure 8
Figure 8
Representative GUS staining pattern of at least four pMeSUS1 promoter-reporter lines. (A) Source leaf (Inlay = Close-up), (B) Sink leaf (Inlay = Close-up), (C) Emerging leaves, (D) Petiole cross-section, (E) Upper stem cross-section, (F) Lower stem cross-section, (G) Storage root cross-section, (H) Fibrous roots, (I) GUS expression levels of three pMeSUS1::GUS lines relative to three pCaMV35S::GUS lines in %. Bars represent mean values with standard deviation (n=4).
Figure 9
Figure 9
Representative GUS staining pattern of four pStPatatin Class I promoter-reporter lines. (A) Source leaf, (B) Sink leaf, (C) Emerging leaves, (D) Petiole cross-section, (E) Upper stem cross-section, (F) Lower stem cross-section, (G) Storage root cross-section, (H) Fibrous roots, (I) GUS expression levels of three pStPatatin : GUS lines relative to three pCaMV35S::GUS lines in %. Bars represent mean values with standard deviation (n=4).
Figure 10
Figure 10
Representative GUS staining pattern of at least four pStB33 promoter-reporter lines. (A) Source leaf, (B) Sink leaf, (C) Emerging leaves, (D) Petiole cross-section, (E) Upper stem cross-section, (F) Lower stem cross-section, (G) Storage root cross-section, (H) Fibrous roots (Inlay = Root tip), (I) GUS expression levels of three pStB33::GUS lines relative to three pCaMV35S::GUS lines in %. Bars represent mean values with standard deviation (n=4).
Figure 11
Figure 11
Representative GUS staining pattern of four pStGBSS1 promoter-reporter lines. (A) Source leaf, (B) Sink leaf, (C) Emerging leaves, (D) Petiole cross-section, (E) Upper stem cross-section, (F) Lower stem cross-section, (G) Storage root cross-section, (H) Fibrous roots (Inlay = Root tip), (I) GUS expression levels of three pStGBSS1:GUS lines relative to three pCaMV35S::GUS lines in %. Bars represent mean values with standard deviation (n=4).
Figure 12
Figure 12
Representative GUS staining pattern of four pMeGPT2 promoter-reporter lines. (A) Source leaf, (B) Sink leaf (Inlay = Close-up), (C) Emerging leaves, (D) Petiole cross-section, (E) Upper stem cross-section, (F) Lower stem cross-section, (G) Storage root cross-section, (H) Fibrous roots, (I) GUS expression levels of three pMeGPT2::GUS lines relative to three pCaMV35S::GUS lines in %. Bars represent mean values with standard deviation (n=4).
Figure 13
Figure 13
Representative GUS staining pattern of at least four pIbSRD1 promoter-reporter lines. (A) Source leaf, (B) Sink leaf, (C) Emerging leaves, (D) Petiole cross-section, (E) Upper stem cross-section, (F) Lower stem cross-section, (G) Storage root cross-section (Inlay = Close-up), (H) Fibrous roots.
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