Germination and early seedling development are critical for successful stand establishment of plants. Following germination, the cotyledons, which are derived from embryonic tissue, emerge from the seed. Arabidopsis seedlings at post-germinative stages are supported mainly by the supply of nutrition from the cotyledons until vegetative leaves emerge and initiate photosynthesis. The switch to autotrophic growth is a significant transition at the post-germinative stage. Here, we provide evidence that down-regulation of SQUAMOSA PROMOTER-BINDING PROTEIN LIKE13 (SPL13) by microRNA156 (miR156) plays an important role in the regulation of the post-germinative switch from the cotyledon stage to the vegetative-leaf stage. Silent mutations created in the SPL13 sequence in the region that is complementary to the miR156 sequence caused the deregulation of the mutant form of SPL13 (mSPL13) mRNA from miR156. Mutant seedlings over-accumulated miRNA-resistant messages and exhibited a delay in the emergence of vegetative leaves compared to wild-type seedlings. The delay was not observed in control transgenic plants expressing non-mutated SPL13, indicating that the phenotype was caused specifically by the silent mutations and deregulation of SPL13 from miR156. Characterization of the SPL13 promoter indicated that this gene is expressed mainly in the hypocotyl and affects leaf primordium development. These results suggest that the repression of SPL13 by miR156 is essential for normal post-germinative growth in Arabidopsis.

MicroRNAs (miRNAs) are involved in developmental programmes of plants, including seed germination and post-germination. Here, we provide evidence that two different miRNA pathways, miR156 and miR172, interact during the post-germination stages in Arabidopsis. Mutant seedlings expressing miR156-resistant SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE13 (mSPL13), which has silent mutations in the miR156 complementary sequence, over-accumulated SPL13 mRNA and exhibited a delay in seedling development. Microarray analysis indicated that SCHNARCHZAPFEN (SNZ), an AP2-like gene targeted by miR172, was downregulated in these mutants. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and miRNA gel blot analyses showed that the MIR172 genes were up-regulated in mSPL13 mutants. These results suggest that the miRNA regulation cascades (miR156 s SPL13 ! miR172 s SNZ) play a critical role during the post-germination developmental stages in Arabidopsis.

). † These authors contributed equally to this work. SUMMARYThe micropylar region of endosperm in a seed, which is adjacent to the radicle tip, is called the 'endosperm cap', and is specifically activated before radicle emergence. This activation of the endosperm cap is a widespread phenomenon among species and is a prerequisite for the completion of germination. To understand the mechanisms of endosperm cap-specific gene expression in tomato seeds, GeneChip analysis was performed. The major groups of endosperm cap-enriched genes were pathogenesis-, cell wall-, and hormone-associated genes. The promoter regions of endosperm cap-enriched genes contained DNA motifs recognized by ethylene response factors (ERFs). The tomato ERF1 (TERF1) and its experimentally verified targets were enriched in the endosperm cap, suggesting an involvement of the ethylene response cascade in this process. The known endosperm cap enzyme endo-b-mannanase is induced by gibberellin (GA), which is thought to be the major hormone inducing endosperm cap-specific genes. The mechanism of endo-bmannanase induction by GA was also investigated using isolated, embryoless seeds. Results suggested that GA might act indirectly on the endosperm cap. We propose that endosperm cap activation is caused by the ethylene response of this tissue, as a consequence of mechanosensing of the increase in embryonic growth potential by GA action.

Coronalon (6-ethyl indanoyl isoleucine), a synthetic jasmonate mimic, is known to regulate levels of transcripts and secondary metabolites that are commonly elicited by methyl jasmonate (MeJA) in a variety of plants. The ability of coronalon and its derivative (In-L-Ile-Me) to elicit MeJA-activated transcriptional and defence responses [nicotine and trypsin proteinase inhibitors (TPIs)] was compared in treated and systemic untreated tissues of wild-type (WT) and NaLOX3-silenced Nicotiana attenuata plants which are unable to activate either local or systemic defence responses. Coronalon and its derivative significantly regulated 71% and 86% of genes up-regulated by MeJA and 53% and 66% of the genes down-regulated by MeJA in the treated leaves, but only 3% and 7% of all regulated genes in untreated, but phylotactically connected, leaves of WT plants. Consistent with their ability to elicit transcriptional responses in treated tissues, coronalon and In-L-Ile-Me increased nicotine and TPIs when applied to the tissues in which these metabolites are produced, namely roots and leaves. However, treating roots elicited TPI activity in leaves in both WT and NaLOX3-silenced plants, suggesting that mimics can be transported apoplastically from roots to leaves in the xylem. This response was lower in NaLOX3-silenced plants, suggesting that the ability of coronalon and In-L-Ile-Me to elicit TPI responses in leaves after root treatments requires intact jasmonic acid (JA) signalling. Treating leaves did not elicit detectable changes in endogenous JA levels but did decrease free salicylic acid contents. It is concluded that coronalon and In-L-Ile-Me elicit jasmonate responses in treated tissues and could be valuable tools for dissecting local and systemic jasmonate signalling networks in plants.

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The objective of the present study was to recognize the molecular background of the accumulation of raffinose family oligosaccharides (RFOs) in pea (Pisum sativum L.) seedlings under osmotic stress conditions. The exposure of 5-day-old pea seedlings to osmotic stress for 48 h created by immersing roots in PEG8000 solution (-1.5 MPa) induced synthesis of galactinol and RFOs (raffinose and stachyose) in the epicotyl and root tissues, but not in cotyledons. After 24 h of recovery, galactinol completely disappeared, raffinose decreased fourfold and stachyose decreased twofold in roots, but increased in epicotyls. The temporary accumulation of RFOs resulted from a dramatic increase in the enzymatic activity and changes in expression of galactinol synthase (PsGolS), raffinose synthase (PsRS) and stachyose synthase (PsSTS) genes. PsGolS was induced by osmotic stress in both epicotyls as well as in roots. PsRS and PsSTS were induced only in epicotyls, but repressed or remained unaffected in roots, respectively. During recovery, the expression and activity of PsGolS, PsRS and PsSTS dramatically decreased. The expression of PsGolS gene, that level of mRNA transcript significantly decreased during recovery and whose promoter region was identified to contain some stress-related regulating elements, seems to play a crucial role in the biosynthesis of RFOs under osmotic stress. Possible signals that may trigger the induction of expression of PsGolS, PsRS and PsSTS genes and accumulation of RFOs in pea seedlings are discussed.

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Key words: lu ci fer ase (LUC); phenylalanine ammo nia-lyase (PAL); ul tra vi o let ra di a tion (UV); β-glucuronidase (GUS) Ab stractThe gene fu sion sys tem was used to study UV light-con trol of PS PAL1 and PS PAL2 genes en cod ing phenylalanine am monia-lyase of pea. The in duc tion of pea PAL pro mot ers was analysed in trans gen ic to bacco plants. Bi nary plasmids (de riv atives of pBI101.2 vec tor) con tain ing 5' reg u la tory frag ments of PS PAL1 and PS PAL2 linked to re porter genes (GUS, LUC) were con structed. The anal y ses were per formed with the use of sin gle con structs (con tain ing one vari ant of PS PAL pro moter and one re porter gene) and dual con structs (con tain ing both PS PAL1 and PS PAL2 pro mot ers con nected with dif fer ent reporter genes). The use of dual con structs en abled the eval u ation of both PS PAL pro mot ers ac tiv ity in the same plant. The anal y ses of in vi tro grown plants have shown that both PAL pro mot ers are strongly in duced in leaves sub jected to UV ra dia tion. In some cases, the UV-stim u lated ex pres sion ex ceeded the ex posed ar eas. This phe nom e non was ob served more of ten in the leaves of plants con tain ing the PS PAL1::GUS than PS PAL2::GUS con struct. Re moval of boxes 2, 4, 5 from PS PAL1 pro moter and de le tion of its 5'end re gion (-339 to -1394) decreases the level of gene ex pres sion but does not elim i nate its re spon sive ness to UV.