The promoter of ZmMRP-1, a maize transfer cell-specific transcriptional activator, is induced at solute exchange surfaces and responds to transport demands

Abstract

Transfer cells have specializations that facilitate the transport of solutes across plant exchange surfaces. ZmMRP-1 is a maize (Zea mays) endosperm transfer cell-specific transcriptional activator that plays a central role in the regulatory pathways controlling transfer cell differentiation and function. The present work investigates the signals controlling the expression of ZmMRP-1 through the production of transgenic lines of maize, Arabidopsis, tobacco and barley containing ZmMRP-1promoter:GUS reporter constructs. The GUS signal predominantly appeared in regions of active transport between source and sink tissues, including nematode-induced feeding structures and at sites of vascular connection between developing organs and the main plant vasculature. In those cases, promoter induction was associated with the initial developmental stages of transport structures. Significantly, transfer cells also differentiated in these regions suggesting that, independent of species, location or morphological features, transfer cells might differentiate in a similar way under the influence of conserved induction signals. In planta and yeast experiments showed that the promoter activity is modulated by carbohydrates, glucose being the most effective inducer.

Fig. 1

a–e The promoter of ZmMRP-1 shows transfer cell specificity in maize. GUS staining pattern of ZmMRP-1prom-GUS transgenic kernels at 10 (a), 14 (b) 20 (c) and 30 (d) days after pollination (DAP). e Basal area of the endosperm from c at a higher magnification. An indigo precipitate, indicating GUS activity, is observed in the transfer cell layer (TCL), which extends at the base of the endosperm, in front of the phloem terminals at the pedicel. En endosperm, Em embryo, Pd pedicel. Bars  1 mm

Fig. 2

a–cZmMRP-1 promoter activity in barley. a GUS staining of an immature barley spike collected at the time of booting. b Detailed view of the rachis after removal of the spikelets. c Cross section of a 5–10 DAP kernel showing GUS signals in the endosperm transfer cell layer (TCL). EC endosperm cavity, CV central vein, En Endosperm. Bar  1 mm

Fig. 3

a–f At germination, the promoter of ZmMRP-1 is active in the endosperm of transgenic Arabidopsis and tobacco. a and b Germinating tobacco seeds stained for GUS; a faint signal is observed over all the endosperm but it is particularly intense in the micropylar endosperm or endosperm cap (Ecap); three seeds showing progressively more developed radicle (R) are shown. c–f Germinating Arabidopsis seeds. c GUS activity is patent in the peripheral endosperm (Pe) in older seedlings. d Transversal section of a seed coat and peripheral endosperm once the plantlet has emerged. e Longitudinal section of a germinating seed. Em embryo. f Enlarged view of the micropylar endosperm (Ecap). Bars  1 mm (a, b), 100 μm (c–f)

Fig. 4

a–f The ZmMRP-1 promoter is regulated by sugars in Arabidopsis seedlings and yeast. GUS activity (a–d) was detected in the inter-cotyledonary nodes (N) and hydathodes (H) in 7–10 day-old transgenic plantlets. The nodes area, squared off in red in a, is shown at higher magnification in b. Arrows point to the vascular nodes formed at both sides of the shoot apical meristem. The left and right nodes are shown at higher magnification in c and d, respectively. Arrowheads in c and d point to the vascular branching and merging areas that concentrate most of the GUS signal. CTv cotyledon vasculature, Sv stem vasculature, LPv leaf primordia vasculature. Bars  1 mm (a), 100 μm (b), 25 μm (c, d). e Transgenic Arabidopsis seedlings (about 200 seeds per assay) were germinated in media containing the indicated concentrations of sugars. GUS activity was measured in the crude protein extracts. At the two sugar concentrations tested, glucose and sucrose (to a lesser extent) induced the promoter. f Promoter activity was measured in yeast clones containing a ZmMRP-1 promoter-lacZ construct. Reporter gene activity was measured after incubation of the cells in media containing, as indicated, glucose, fructose, sucrose, mannitol or glycerol (cont). Glucose and fructose strongly induced the promoter. Mean values ± SD (e, f)

Fig. 5

a–d Expression of ZmMRP-1 at the young branching points of mature plants. a A branching point in the stem of Arabidopsis; the GUS signal is concentrated on the dorsal side of the vasculature. b Mid section of a tobacco stem at the emerging point of two lateral leaves and branches; areas squared off in red in b and c indicate the regions used to produce the higher magnification images in c and d, respectively. The most intense GUS signal concentrates in the transformed xylem parenchyma cells above the vasculature. Bars  1 mm (a, b, c), 100 μm (d)

Fig. 6

a–d The ZmMRP-1 promoter is active at the base of reproductive structures. a GUS expression at the base of flowers and fruits in Arabidopsis; the signal intensity increases from flower budding (FB) to reach a maximum at the pedicel of the siliques at the mid-maturation stage (MMS); it then decreases as the fruit enters the seed filling phase. b GUS signal at the base of a tobacco flower (arrow). c The signal is most intense at the pedicel (Pd), the vasculature nodes (VN) and the placental area (Pl) under the seeds (S) of a pollinated tobacco flower at mid-maturation phase. d The GUS signal disappears from the pedicel of an emasculated non-pollinated flower (NP) but reaches normal levels (arrow) in an emasculated manually-pollinated flower (P) emerging at the same branching point

Fig. 7

a–d The promoter of ZmMRP-1 is active in the early developmental stages of the feeding structures induced by nematode infection. a A transgenic Arabidopsis root 5 days after inoculation with Meloidogyne javanica larvae showing the GUS signal in the gall (arrow). b A gall 10 days after inoculation. c A gall 15 days after inoculation. d A gall 20 days after inoculation