Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa

Nucleotide sequences alignment between (a) M. sativa SPL2/3/4 and Arabidopsis SPL3/4/5; (b) MsSPL9 and AtSPL9, respectively. (TIF 3681 kb

MsSPL9 Modulates Nodulation under Nitrate Sufficiency Condition in Medicago sativa

Nodulation in Leguminous spp. is induced by common environmental cues, such as low nitrogen availability conditions, in the presence of the specific Rhizobium spp. in the rhizosphere. Medicago sativa (alfalfa) is an important nitrogen-fixing forage crop that is widely cultivated around the world and relied upon as a staple source of forage in livestock feed. Although alfalfa’s relationship with these bacteria is one of the most efficient between rhizobia and legume plants, breeding for nitrogen-related traits in this crop has received little attention. In this report, we investigate the role of Squamosa-Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in nodulation in alfalfa. Transgenic alfalfa plants with SPL9-silenced (SPL9-RNAi) and overexpressed (35S::SPL9) were compared to wild-type (WT) alfalfa for phenotypic changes in nodulation in the presence and absence of nitrogen. Phenotypic analyses showed that silencing of MsSPL9 in alfalfa caused an increase in the number of nodules. Moreover, the characterization of phenotypic and molecular parameters revealed that MsSPL9 regulates nodulation under a high concentration of nitrate (10 mM KNO3 ) by regulating the transcription levels of the nitrate-responsive genes Nitrate Reductase1 (NR1), NR2, Nitrate transporter 2.5 (NRT2.5), and a shoot-controlled autoregulation of nodulation (AON) gene, Super numeric nodules (SUNN). While MsSPL9–overexpressing transgenic plants have dramatically increased transcript levels of SUNN, NR1, NR2, and NRT2.5, reducing MsSPL9 caused downregulation of these genes and displayed a nitrogen-starved phenotype, as downregulation of the MsSPL9 transcript levels caused a nitrate-tolerant nodulation phenotype. Taken together, our results suggest that MsSPL9 regulates nodulation in alfalfa in response to nitrate

Deciphering the role of SPL12 and AGL6 from a genetic module that functions in nodulation and root regeneration in Medicago sativa.

Our results show that SPL12 plays a crucial role in regulating nodule development in Medicago sativa L. (alfalfa), and that AGL6 is targeted and downregulated by SPL12. Root architecture in plants is critical because of its role in controlling nutrient cycling, water use efficiency and response to biotic and abiotic stress factors. The small RNA, microRNA156 (miR156), is highly conserved in plants, where it functions by silencing a group of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. We previously showed that transgenic Medicago sativa (alfalfa) plants overexpressing miR156 display increased nodulation, improved nitrogen fixation and enhanced root regenerative capacity during vegetative propagation. In alfalfa, transcripts of eleven SPLs, including SPL12, are targeted for cleavage by miR156. In this study, we characterized the role of SPL12 in root architecture and nodulation by investigating the transcriptomic and phenotypic changes associated with altered transcript levels of SPL12, and by determining SPL12 regulatory targets using SPL12-silencing and -overexpressing alfalfa plants. Phenotypic analyses showed that silencing of SPL12 in alfalfa caused an increase in root regeneration, nodulation, and nitrogen fixation. In addition, AGL6 which encodes AGAMOUS-like MADS box transcription factor, was identified as being directly targeted for silencing by SPL12, based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays. Taken together, our results suggest that SPL12 and AGL6 form a genetic module that regulates root development and nodulation in alfalfa

Enhanced accumulation of root hydrogen peroxide is associated with reduced antioxidant enzymes under isoosmotic NaCl and Na2SO4 salinities

The inhibitory effect of salt stress on lettuce is one of the main reasons for the reduction of plant growth and crop productivity. In the present study, the response of two lettuce varieties Verte and Romaine to isoosmotic NaCl and Na2SO4 treatments were examined. Both varieties were grown in pots containing nutrient Hoagland solution with or without 100 mM NaCl or 77 mM Na2SO4. Relative growth rate (RGR), hydraulic parameters, root ion content, proline and several antioxidant activities in roots were measured after 12 days of treatment. After prolonged exposure to salt stress, relative growth rate and water content of lettuce significantly decreased. Roots accumulated high level of Na+ under both salts, whereas the accumulation of K+ and Ca2+ decreased. High level of Na+ inside the cells inhibited the K+ uptake and resulted in increased K+/Na+ ratio. In addition, salt stress also caused an increase in the accumulation of proline. This result suggests that proline may play a crucial role in protecting lettuce under salt stress especially in response to Na2SO4 treatment. Membrane damage estimated by electrolyte leakage (EL) increased especially in response to Na2SO4 treatment in both varieties, but Verte had significantly lower EL relative to Romaine under 100 mM NaCl. A reduction in the activities of CAT in both varieties under 100 mM, and GPX activity in Verte under Na2SO4 treatment coincided with an increase in H2O2 level, indicative of cellular damage and a general depression of the antioxidant enzymatic system in lettuce roots.Keywords: Lettuce, NaCl, Na2SO4, RGR, mineral nutrition, antioxidant activities, prolin

Corrigendum: miR156/SPL10 Modulates Lateral Root Development, Branching and Leaf Morphology in Arabidopsis by Silencing AGAMOUS-LIKE 79

The developmental functions of miR156-SPL regulatory network have been extensively studied in Arabidopsis, but the downstream genes regulated by each SPL have not been well characterized. In this study, Next Generation Sequencing-based transcriptome analysis was performed on roots of wild type (WT) and miR156 overexpression (miR156OE) plants. One of the SPL genes, SPL10, which represses lateral root growth in Arabidopsis, was significantly downregulated in miR156OE plants. A transcription factor, AGAMOUS-like MADS box protein 79 (AGL79), was also significantly downregulated in the miR156OE plants, but was upregulated in the SPL10 overexpression (SPL10OE) Arabidopsis plants. In addition, SPL10 was found to bind to the core consensus SPL binding sequences in AGL79 gene. Moreover, analyses of complementation lines revealed a linear relationship between SPL10 and AGL79 in regulating Arabidopsis plant development. In addition, it was observed that plant phenotypes are AGL79 dose-dependent, with higher expression causing narrow leaf shape, less number of leaves and early flowering time, whereas relatively lower AGL79 overexpression produce plants with more rosette leaves and more lateral branches. Our findings revealed direct binding of SPL10 to AGL79 promoter, which further suggests a role for miR156/SPL10 module in plant lateral root growth by directly regulating AGL79

Combined effect of hormonal priming and salt treatments on germination percentage and antioxidant activities in lettuce seedlings

Hormonal priming is a pre-sowing treatment that improves seed germination performance and stress tolerance. To understand the physiology of hormonal priming and its association with post priming stress tolerance, we investigated the effect of hormonal priming with increasing gibberellic acid (GA3) concentrations (0, 3, 4.5 and 6 mM) on seedling growth and antioxidant system in lettuce. Germination percentage was higher in lettuce seedlings derived from primed seeds. Radicle and hypocotyl length and dry weight were reduced by salt treatment to a greater extent in non-primed than in primed seeds. Hormonal priming with 4.5 mM GA3 induced the most dramatic decreases in electrolyte leakage (EL) and malondialdehyde (MDA) levels. NaCl increased catalase (CAT) activity in primed and non-primed seeds. The total ascorbate level remained constant in both primed and non-primed seeds under NaCl constraint. These results suggest that hormonal priming might have increased the salt tolerance of lettuce seeds through enhancing the activities of antioxidant enzymes and reducing the membrane damage as estimated using EL and MDA biomarkers.Key words: Ascorbate, germination, hormonal priming, lettuce, salinity