MicroRNA156 (miR156) functions in maintaining the juvenile phase in plants. However, the mobility of this microRNA has not been demonstrated. So far, only three microRNAs, miR399, miR395, and miR172, have been shown to be mobile. We demonstrate here that miR156 is a potential graft-transmissible signal that affects plant architecture and tuberization in potato (Solanum tuberosum). Under tuber-noninductive (long-day) conditions, miR156 shows higher abundance in leaves and stems, whereas an increase in abundance of miR156 has been observed in stolons under tuber-inductive (short-day) conditions, indicative of a photoperiodic control. Detection of miR156 in phloem cells of wild-type plants and mobility assays in heterografts suggest that miR156 is a graft-transmissible signal. This movement was correlated with changes in leaf morphology and longer trichomes in leaves. Overexpression of miR156 in potato caused a drastic phenotype resulting in altered plant architecture and reduced tuber yield. miR156 overexpression plants also exhibited altered levels of cytokinin and strigolactone along with increased levels of LONELY GUY1 and StCyclin D3.1 transcripts as compared with wild-type plants. RNA ligase-mediated rapid amplification of complementary DNA ends analysis validated SQUAMOSA PROMOTER BINDING-LIKE3 (StSPL3), StSPL6, StSPL9, StSPL13, and StLIGULELESS1 as targets of miR156. Gel-shift assays indicate the regulation of miR172 by miR156 through StSPL9. miR156-resistant SPL9 overexpression lines exhibited increased miR172 levels under a short-day photoperiod, supporting miR172 regulation via the miR156-SPL9 module. Overall, our results strongly suggest that miR156 is a phloem-mobile signal regulating potato development.

Potato Homeobox1 (POTH1) is a Knotted1-like transcription factor from the Three Amino Acid Loop Extension (TALE) superfamily that is involved in numerous aspects of development in potato (Solanum tuberosum L). POTH1 interacts with its protein partner, StBEL5, to facilitate binding to specific target genes to modulate hormone levels, mediate leaf architecture, and enhance tuber formation. In this study, promoter analyses show that the upstream sequence of POTH1 drives β-glucuronidase activity in response to light and in association with phloem cells in both petioles and stems. Because POTH1 transcripts have previously been detected in phloem cells, long-distance movement of its mRNA was tested. Using RT-PCR and transgenic potato lines over-expressing POTH1, in vitro micrografts demonstrated unilateral movement of POTH1 RNA in a rootward direction. Movement across a graft union into leaves from newly arising axillary shoots and roots of wild type stocks was verified using soil-grown tobacco heterografts. Leaves from the wild type stock containing the mobile POTH1 RNA exhibited a reduction in leaf size relative to leaves from wild type grafts. Both untranslated regions of POTH1 when fused to an expression marker β-glucuronidase, repressed its translation in tobacco protoplasts. RNA/protein binding assays demonstrated that the UTRs of POTH1 bind to two RNA-binding proteins, a polypyrimidine tract-binding protein and an alba-domain type. Conserved glycerol-responsive elements (GRE), specific to alba-domain interaction, are duplicated in both the 5' and 3' untranslated regions of POTH1. These results suggest that POTH1 functions as a mobile signal in regulating development.

Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (β-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development.

Phytochromes comprise a small family of photoreceptors with which plants gather environmental information that they use to make developmental decisions, from germination to photomorphogenesis to fruit development. Most phytochromes are activated by red light and de-activated by far-red light, but phytochrome A (phyA) is responsive to both and plays an important role during the well-studied transition of seedlings from dark to light growth. The role of phytochromes during skotomorphogenesis (dark development) prior to reaching light, however, has received considerably less attention although previous studies have suggested that phytochrome must play a role even in the dark. We profiled proteomic and transcriptomic seedling responses in tomato during the transition from dark to light growth and found that phyA participates in the regulation of carbon flux through major primary metabolic pathways, such as glycolysis, beta-oxidation, and the tricarboxylic acid (TCA) cycle. Additionally, phyA is involved in the attenuation of root growth soon after reaching light, possibly via control of sucrose allocation throughout the seedling by fine-tuning the expression levels of several sucrose transporters of the SWEET gene family even before the seedling reaches the light. Presumably, by participating in the control of major metabolic pathways, phyA sets the stage for photomorphogenesis for the dark grown seedling in anticipation of light.