Three members of Medicago truncatula ST family are ubiquitous during development and modulated by nutritional status (MtST1) and dehydration (MtST2 and MtST3)

Abstract

Background: ShooT specific/Specific Tissue (ST) belong to a protein family of unknown function characterized by the DUF2775 domain and produced in specific taxonomic plant families, mainly Fabaceae and Asteraceae, with the Medicago truncatula ST family being the largest. The putative roles proposed for this family are cell elongation, biotic interactions, abiotic stress and N reserve. The aim of this work was to go deeper into the role of three M. truncatula ST proteins, namely ST1, ST2 and ST3. Our starting hypothesis was that each member of the family could perform a specific role, and hence, each ST gene would be subjected to a different type of regulation.

Results: The search for cis-acting regulatory elements (CREs) in silico in pST1, pST2 and pST3 promoters showed prevalence of tissue/organ specific motifs, especially root- and seed-specific ones. Light, hormone, biotic and abiotic related motifs were also present. None of these pSTs showed the same combination of CREs, or presented the same activity pattern. In general, pST activity was associated with the vascular cylinder, mainly in roots. Promoter activation was highly specific and dissimilar during reproductive development. The ST1, ST2 and ST3 transcripts accumulated in most of the organs and developmental stages analysed - decreasing with age - and expression was higher in the roots than in the aerial parts and more abundant in light-grown plants. The effect of the different treatments on transcript accumulation indicated that ST1 behaved differently from ST2 and ST3, mainly in response to several hormones and dehydration treatments (NaCl or mannitol), upon which ST1 transcript levels decreased and ST2 and ST3 levels increased. Finally, the ST1 protein was located in the cell wall whereas ST2 and ST3 were present both in the cytoplasm and in the cell wall.

Conclusions: The ST proteins studied are ubiquitous proteins that could perform distinct/complementary roles in plant biology as they are encoded by differentially regulated genes. Based on these differences we have established two functional groups among the three STs. ST1 would participate in processes affected by nutritional status, while ST2 and ST3 seem to act when plants are challenged with abiotic stresses related to water stress and in physiologically controlled desiccation processes such as the seed maturation.

Keywords: Abiotic stress; DUF2775; Development; Medicago truncatula; ST protein; cis-acting regulatory elements.

Fig. 1

CREs present in pST1, pST2 and pST3 according to in silico predictions using PLACE and PlantCARE. a Percentage of CREs belonging to the different categories established in this work. b Position of CREs in the tissue specific and light categories. c Position of CREs in the hormone, biotic and abiotic categories. Highly abundant CREs (ARRIAT, CAATBOX, GATABOX, GTGANTG10, DOFCOREZM) were not represented in order to simplify the figure. bp: base pairs upstream ATG

Fig. 2

Activity of Medicago truncatula pST1, pST2 and pST3 in Arabidopsis thaliana seedlings. Histochemical GUS staining in transgenic pST::GUS seedlings at 3- (a, b) and 10-d-old (c, d) grown in darkness (a, c) or light (b, d). Numbers refer to the corresponding ST gene. c, cotyledon; h, hypocotyl; l, leaf; r, root; ra, root apex. The blue colour and the arrows indicate the zones with GUS activity driven by pST

Fig. 3

Activity of Medicago truncatula pST1, pST2 and pST3 in vegetative organs of Arabidopsis thaliana plants. Histochemical GUS staining in vegetative organs (a, roots; b-d, aerial parts) of 10- to 24-d-old pST::GUS transgenic plants. Numbers refer to the corresponding ST gene. c, cotyledon; er, emerging root; fs, floral stem; h, hypocotyl; l, leaf; r, root; ra, root apex; s, stoma. The blue colour and the arrows indicate the zones with GUS activity driven by pST

Fig. 4

Activity of Medicago truncatula pST1, pST2 and pST3 in reproductive organs of Arabidopsis thaliana. Histochemical GUS staining during reproductive development of arabidopsis pST::GUS plants (a to g sorted according to developmental stage). Numbers refer to the corresponding ST gene. a, anther; e, embryo; f, filament; fb, floral bud; fl, flower; o, ovary; p, pollen; s, seed; sg, stigma; si, silique; st, style. The blue colour and the arrows indicate the zones with GUS activity driven by pST

Fig. 5

sqRT-PCR of Medicago truncatula ST1, ST2 and ST3 transcripts along development. a Transcript accumulation in 24 hpi seeds and in 3-, 6- and 10-d-old dark-grown seedlings (roots and aerial parts). b Transcript accumulation in 3-, 6- and 10-d-old light-grown seedlings (roots and aerial parts). c Transcript accumulation in several organs of adult plants: root from 30-d-old plants; leaves, flowers and pods in 3 developmental stages; and green seed. The different development stages of leaves were: Leaf 1, small, closed folioles without spot; Leaf 2, fully expanded leaves, folioles with spot smaller than 1 cm; and Leaf 3, fully expanded leaves, folioles with spot bigger than 1 cm. The different developmental stages of flowers were: Flower 1, green immature with sepals covering the organs; Flower 2, closed yellow flower two days before anthesis; and Flower 3, at anthesis. The different developmental stage of pods were: Pod 1, early pod with a complete spiral; Pod 2, pod with 5 complete spirals and initial spines with embryo at globular state; and Pod 3, pod with 6 complete spirals and mature spines with embryo at heart state. Green seeds are seeds collected from 24 to 26 d after pollination. c1 Detail of bars between 0 and 50 units showed in c (red square). Transcript accumulation was measured relative to ubiquitin as indicated in the Methods section. Scale units express normalized and integrated absorbance (nIA). More detailed description of plant material is included in Additional file 3. Statistical analyses were performed separately for each ST gene as stated in the Methods section. The different letters on the tops of the histograms indicate a statistically significant difference of p < 0.05

Fig. 6

sqRT-PCR of ST1, ST2 and ST3 transcripts in Medicago truncatula seedlings under different treatments. a Transcript accumulation in untreated 7-d-old seedlings (control) and 7-d-old seedlings exposed during the last 24 h to indolacetic acid (IAA, 10 μM); benzylaminopurine (CK, 10 μM); giberellic acid (GA, 100 μM); epibrassinolide (BL, 10 μM) and strigolactone GR24 (SL, 10 μM). b Transcript accumulation in untreated 7-d-old seedlings (control) and 7-d-old seedlings exposed during the last 24 h to abscisic acid (ABA, 100 μM); sodium chloride (NaCl, 150 mM); mannitol (250 mM); N starvation (−N) plants where kept for 7 days in Fahräeus without N supplement and Pi starvation (−Pi) plants where kept for 7 days in Fahräeus-N without phosphate. c Transcript accumulation in plants exposed at different temperatures: 25 °C (control), −4 °C and 37 °C in darkness for the last 12 h in 7-d-old seedlings. d: Transcript accumulation in untreated 7-d-old seedlings (control) and 7-d-old seedlings exposed during the last 24 h to ethephon (ET, 1 mM); methyl jasmonate (MeJA, 100 μM); salicylic acid (SA, 1 mM); as well as a mixture of MeJA with one of the following: ET (MeJA + ET); SA (MeJA + SA) and GA (MeJA + GA) at the same concentrations as when individually applied. Wounding was performed in one foliole by transversally cutting the middle vein. Transcript accumulation was measured relative to ubiquitin as indicated in the Methods section. Scale units express normalized and integrated absorbance (nIA). Statistical analyses were conducted as stated in the Methods section considering 3 levels of significance: *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 7

Subcellular location of Medicago truncatula ST1, ST2 and ST3 proteins in Arabidopsis thaliana. Confocal microscopy images of roots from Arabidopsis p35S::ST-GFP plants (ST1, ST2 and ST3) mounted in water or in 0.6 M mannitol. First column (GFP) show green fluorescence from ST-GFP fusion proteins, second column (PI) red fluorescence for propidium iodide stained cell walls and third column (GFP + PI) both channels merged with DIC channel. Bars = 25 μm