Symplasmic phloem unloading and radial post-phloem transport via vascular rays in tuberous roots of Manihot esculenta

Abstract

Cassava (Manihot esculenta) is one of the most important staple food crops worldwide. Its starchy tuberous roots supply over 800 million people with carbohydrates. Yet, surprisingly little is known about the processes involved in filling of those vital storage organs. A better understanding of cassava carbohydrate allocation and starch storage is key to improving storage root yield. Here, we studied cassava morphology and phloem sap flow from source to sink using transgenic pAtSUC2::GFP plants, the phloem tracers esculin and 5(6)-carboxyfluorescein diacetate, as well as several staining techniques. We show that cassava performs apoplasmic phloem loading in source leaves and symplasmic unloading into phloem parenchyma cells of tuberous roots. We demonstrate that vascular rays play an important role in radial transport from the phloem to xylem parenchyma cells in tuberous roots. Furthermore, enzymatic and proteomic measurements of storage root tissues confirmed high abundance and activity of enzymes involved in the sucrose synthase-mediated pathway and indicated that starch is stored most efficiently in the outer xylem layers of tuberous roots. Our findings form the basis for biotechnological approaches aimed at improved phloem loading and enhanced carbohydrate allocation and storage in order to increase tuberous root yield of cassava.

Keywords: Apoplast; CFDA; SUC2; cassava; esculin; morphology; phloem; ray; starch; symplast.

Fig. 1.

Phloem and xylem cells in tuberous roots of cassava are connected by specialized cell files. (A) Overview of the cassava rootstock. (B) Cross-section of fibrous root. (C) Cross-section of developing tuberous root. Inset shows the breakage of endodermis/pericycle and a structure resembling a newly formed vascular ray. (D) Cross-section of tuberous root neck region. Inset shows an enlarged example of a vascular ray. (E) Cross-section of tuberous root. (B–E) All sections were stained with 0.1% toluidine blue. Ex, exodermis; P, phloem; PC, procambium; Per, periderm; VC, vascular cambium; X, xylem. Arrowheads indicate vascular rays.

Fig. 2.

Free GFP can diffuse through parenchymatic vascular rays in cassava tuberous roots. (A) Confocal image of WT petiole cross-section. (B, C) Confocal image of pAtSUC2::GFP petiole cross-section. Arrowheads indicate cells with strongest GFP signal. (D) Confocal image of WT upper stem cross-section. (E, F) Confocal image of pAtSUC2::GFP upper stem cross-section. Arrowheads indicate cells with strongest GFP signal. (G) Confocal image of WT tuberous root cross-section. (H) Confocal image of pAtSUC2::GFP tuberous root cross-section. Arrowheads indicate vascular rays connecting phloem and xylem. (I) Maximum projection of pAtSUC2::GFP tuberous root longitudinal section. Arrowheads indicate sieve element-shaped cell files with strongest GFP signal. (A–I) All sections were counterstained with propidium iodide (magenta). All sections were merged with bright field. GFP fluorescence in green. P, phloem; VC, vascular cambium; X, xylem.

Fig. 3.

Transgenic pAtSUC2::GFP cassava plants produce GFP in the shoot and translocate it to the roots. Upper lane shows GFP RT-PCR results in different tissues of WT and pAtSUC2::GFP plants. Second lane shows ubiquitin RT-PCR results in different tissues of WT and pAtSUC2::GFP plants. Third lane shows detected GFP protein in different tissues of WT and pAtSUC2::GFP plants via immunoblot using an anti-GFP antibody. Bottom lane shows the loading control in the form of a Coomassie gel with equally loaded protein extract amounts compared with the immunoblot.

Fig. 4.

Detection of GFP in rootstocks of grafted pAtSUC2::GFP shoots onto WT rootstocks proves movement of GFP from shoot into tuberous roots. (A) Confocal image of WT tuberous root cross-section. (B) pAtSUC2::GFP scion grafted onto WT rootstock. Arrowhead indicates grafting region. (C, D) Confocal image of tuberous root cross-section of pAtSUC2::GFP shoot × WT rootstock grafted plants. Arrowheads indicate vascular rays. (A, C, D) All sections were counterstained with propidium iodide (magenta). All sections are shown with bright field. GFP fluorescence in green. P, phloem; VC, vascular cambium; X, xylem.

Fig. 5.

Esculin indicates apoplasmic phloem loading. (A) Autofluorescence in WT petiole. Xylem autofluorescence in white/blue, chlorophyll in red. (B) Fluorescence in petiole cross-section of esculin-loaded WT plant. Esculin in bright light blue, chlorophyll in red. (C, D) Fluorescence in petiole longitudinal section of esculin-loaded WT plant. Esculin in bright light blue, chlorophyll in red. (E) Confocal image of petiole cross-section of esculin-loaded WT plant. Arrowheads indicate sieve plates stained with aniline blue. Esculin fluorescence is displayed in green. (F) Fluorescence in tuberous root WT cross-section. Xylem autofluorescence in white/light blue. Arrowheads indicate vascular rays. (G) Fluorescence in tuberous root cross-sections of esculin-loaded WT plant. Xylem autofluorescence in white/light blue, esculin fluorescence in blue. Arrowheads indicate vascular rays. (A–G) P, phloem; SE, sieve element; VC, vascular cambium; X, xylem.

Fig. 6.

CF monitoring and Lugol staining highlights the importance of vascular rays for carbohydrate transport and storage in tuberous roots. (A) Confocal image with bright field of WT tuberous root cross-section. Propidium iodide in red. (B) Tile-scan picture section of tuberous root cross-section of CFDA-loaded WT plant. Propidium iodide in red, carboxyfluorescein in green. (C) Confocal image with bright field of tuberous root cross-section of CFDA-loaded WT plant. Carboxyfluorescein in green. (D–F) Starch appearing in black/brown after Lugol staining in cross-section of WT tuberous root. Iodide staining in black/brown. (A–F) Arrowheads indicate vascular rays connecting phloem and xylem. P, phloem; VC, vascular cambium; X, xylem.

Fig. 7.

GFP and CF are confined to the fibrous root phloem cell, while esculin can be unloaded. (A) Confocal image of a WT fibrous root cross-section. (B) Confocal image of a pAtSUC2::GFP fibrous root cross-section. (C) Confocal image towards the tip of a fibrous root cross-section of a CFDA-loaded WT plant. (D) Confocal image of a fibrous root cross-section of a CFDA-loaded WT plant. (E, F) Confocal image of a fibrous root longitudinal-section of a CFDA-loaded WT plant. (G, H) Fluorescence in fibrous root cross-sections of esculin-loaded plants. Esculin fluorescence in blue, within the cortex area surrounding the vascular cylinder. (I) Fluorescence in fibrous root longitudinal sections of esculin-loaded plants. Esculin fluorescence in blue, within the cortex area outside the vascular cylinder. (A–F) All sections were counterstained with propidium iodide (magenta). All sections are shown with bright field. GFP and CF fluorescence in green. (A–I) Cor, cortex; P, phloem; PC, procambium; X, xylem.

Fig. 8.

Tuberous roots of cassava mainly use the sucrolytic pathway for starch synthesis and starch is stored most efficiently in the outer xylem. (A) Overview of the sampled tuberous root regions of field-grown cassava genotype Z010116. P, phloem; X₁–X₆, xylem fractions 1–6; X₁, outermost xylem fraction; X₆, most central xylem fraction. (B) Measured sucrose synthase (SUS) enzyme activity in the phloem and xylem fractions. (C) Measured cell wall invertase (CWI) enzyme activity in the phloem and xylem fractions. (D) Starch concentration in the phloem and xylem fractions. (E) Overview of the sampled tuberous root regions of field-grown cassava genotype I050128 for comparative proteomics. IX, inner xylem; OX, outer xylem; P, phloem and outer root parts. (F) Relative SUS protein levels in phloem, outer xylem and inner xylem samples. Mean intensity determined for each fraction was set to 1. (G) Relative ADP-glucose pyrophosphorylase protein levels in phloem, outer xylem and inner xylem samples. Mean intensity determined for each fraction was set to 1. APL, ADP-glucose pyrophosphorylase large subunit; APS, ADP-glucose pyrophosphorylase small subunit. (H) Fructose-6-phosphate concentration in the phloem and xylem fractions. (I) Glucose-6-phosphate concentration in the phloem and xylem fractions. (J) Glucose-1-phosphate concentration in the phloem and xylem fractions. (K) ATP concentration in the phloem and xylem fractions. (B–D, H–K) Diagrams show the mean of data from ≥4 biological replicates together with the standard error. (F, G) Diagrams show the mean of data from three biological replicates together with the standard error.

Fig. 9.

Model of cassava assimilate transport and potential targets for biotechnological yield improvement. Sucrose produced via photosynthesis is transported from the mesophyll cells into the apoplast. Sucrose carriers of the SUC/SUT-family transport sucrose from the apoplast into the phloem companion cell symplast. Sucrose can diffuse through pore plasmodesmal units into the sieve elements for long-distance transport. Sucrose is unloaded via companion cells into the phloem parenchyma cells. Sucrose can diffuse within the phloem and outer parts of the tuberous root; however, the bulk of sucrose diffuses from the phloem towards the xylem parenchyma via vascular rays and is stored as starch. In contrast to the cambial ray initials that are symplasmically connected to phloem and xylem, the fusiform cambial initials are isolated from the tuberous root symplast and likely have to be supplied with sugars through the activity of cell wall invertases and monosaccharide carrier proteins. Potential targets for biotechnological yield improvement are outlined in red and the number corresponds to the following references: 1: Dasgupta et al. (2014), Wang et al. (2015); 2: Jin et al. (2009), Ruan et al. (2010); 3: Jang et al. (2015), Immanen et al. (2016); 4: Ihemere et al. (2006); 5: Vigeolas et al. (2011). H⁺, proton; Suc, sucrose; SUS, sucrose synthase; UDP-Glc, UDP-glucose.