Predominantly symplastic phloem unloading of photosynthates maintains efficient starch accumulation in the cassava storage roots (Manihot esculenta Crantz)

Abstract

Background: Cassava (Manihot esculenta Crantz) efficiently accumulates starch in its storage roots. However, how photosynthates are transported from the leaves to the phloem (especially how they are unloaded into parenchymal cells of storage roots) remains unclear.

Results: Here, we investigated the sucrose unloading pattern and its impact on cassava storage root development using microstructural and physiological analyses, namely, carboxyfluorescein (CF) and C¹⁴ isotope tracing. The expression profiling of genes involved in symplastic and apoplastic transport was performed, which included enzyme activity, protein gel blot analysis, and transcriptome sequencing analyses. These finding showed that carbohydrates are transported mainly in the form of sucrose, and more than 54.6% was present in the stem phloem. Sucrose was predominantly unloaded symplastically from the phloem into storage roots; in addition, there was a shift from apoplastic to symplastic unloading accompanied by the onset of root swelling. Statistical data on the microstructures indicated an enrichment of plasmodesmata within sieve, companion, and parenchyma cells in the developing storage roots of a cultivar but not in a wild ancestor. Tracing tests with CF verified the existence of a symplastic channel, and [¹⁴C] Suc demonstrated that sucrose could rapidly diffuse into root parenchyma cells from phloem cells. The relatively high expression of genes encoding sucrose synthase and associated proteins appeared in the middle and late stages of storage roots but not in primary fibrous roots, or secondary fibrous roots. The inverse expression pattern of sucrose transporters, cell wall acid invertase, and soluble acid invertase in these corresponding organs supported the presence of a symplastic sucrose unloading pathway. The transcription profile of genes involved in symplastic unloading and their significantly positive correlation with the starch yield at the population level confirmed that symplastic sucrose transport is vitally important in the development of cassava storage roots.

Conclusions: In this study, we revealed that the cassava storage root phloem sucrose unloading pattern was predominantly a symplastic unloading pattern. This pattern is essential for efficient starch accumulation in high-yielding varieties compared with low-yielding wild ancestors.

Keywords: Carbohydrates; Cassava; Starch yield; Storage root; Symplastic unloading.

Fig. 1

The morphological characteristic, biomass accumulation, substances transported in cassava root. a Primary fibrous root. b Secondary fibrous root. c Storage root. d The curves of biomass accumulation of cassava storage root in the three growth stages. The values of the fresh and dry weight are the means of three replicates ±SD. e HPLC–ELSD chromatograms of the cassava phloem exudates. The yield curve in d was performed by Micro software Excel 2016. All of them were with cropped edges and adjusted position using Adobe Photoshop CS6.0 software. Bars in a–c, 1 cm

Fig. 2

Ultrastructure of phloem cells in storage root of cassava showing highly dense plasmodesmata. a–c, d–f, g–i, j–l Phloem cells in the primary fibrous root and storage root at the early, middle, and late stages, respectively. a, g, j a partial enlargement of b, h and k respectively. c, f, i, l Arrows indicated plasmodesmata between two PCs in the primary fibrous root and storage root at the early, middle, and late stages, respectively. d, e Plasmodesmata between a PC and adjacent CC cell, between SE and CC in early storage roots respectively. h, k A larger scope of the transverse section of the phloem cell group in the storage root at the middle and late stages. CW in k indicates a thickened cell wall. All of them were with cropped edges and adjusted position using Adobe Photoshop CS6.0 software. Abbreviations, SE, sieve element; CC, companion cell; PC, parenchyma cell; CW, cell wall

Fig. 3

Carboxyfluorescein (CF) and [¹⁴C] Suc tracing support a predominantly symplasmic phloem unloading pathway in the storage root of cassava. a, b Transverse section anatomy of the storage root. c–f5 CLSM imaging of CF unloading during cassava root development. c–c3 Primary fibrous root. c2 Bright–field microscopy. c3 405nm excitation wave and bright–field microscopy. d–d5 Early stage storage root. e–e5 Middle stage storage root. f–f5 Late stage storage root. c1, d3–d5, e3–e5, f3–f5 Bright–fiield and 488 nm excitation wave microscopy. g CF fluorescence intensity of different root develop–mental stages. h The difference in the distribution density of [¹⁴C] Suc in cross sections of developmental roots at the three stages. The arrows indicate the vascular cambium. All of them with cropped edges and adjusted position using Adobe Photoshop CS6.0 software. Abbreviations, E, Early stage of storage root; M, Middle stage of storage root; L, Late stage of storage root; FBR, Primary fibrous root. Bar = 5 cm in a; Bar = 1 μm in b; Bar = 200 nm in c-f5; Bar = 1 cm in h

Fig. 4

The expression and enzymatic activities of genes involved in post phloem unloading in the storage root of cassava. a, b Relative expression difference of CWI and SuSy measured by Q–PCR. c Heat map comparisons of differential gene expression of CWIs, SuSys and SUTs. d The expression profiling of SUTs (SUT1, SUT2 and SUT4) in leaves and storage roots measured by Q–PCR. e The expressed protein levels of SuSy, CWI and SAI assessed by immunoblotting. f Enzymatic activities of SuSy (triangles), CWI (squares) and SAI (circles). The column chart in a, b, d and the curve chart in f were performed by Micro software Excel 2016. All of them were with cropped edges and adjusted position using Adobe Photoshop CS6.0 software. E, Early stage of storage root; M, Middle stage of storage root; L, Late stage of storage root; PFR, Primary fibrous root and SFR, Secondary fibrous root; a, b and c denote significantly different levels at P < 0.05

Fig. 5

Subcellular location of CWI presented its enrichment in early fibrous roots using immunogold particle labeling. a–d Immunogold labeling of CWI in primary fibrous roots with an antibody directed against cassava acid invertases. e–h Immunogold labeling of CWI in storage roots with an antibody directed against cassava acid invertases. All of them were with cropped edges and adjusted position using Adobe Photoshop CS6.0 software

Fig. 6

A model of sucrose symplastic phloem unloading in the storage root of cassava. Long–distance transported SUC in the SE enters the CC and PC predominantly through plasmodesmata, with the exception of a few SUCs that are transported by SUTs. These SUC molecules are primarily catabolized by SuSy into fructose and nucleoside diphosphate–glucose (UDPG), which enter starch synthesis in the amyloplast. Occasionally, SUC can be stored temporarily in vacuoles by SUT or HT in a hexose form. This structured energy-saving model accounts for the highly efficient starch accumulation observed in cassava storage roots. This model diagram was performed by Micro software Powerpoint 2016