SummaryPlants contain two genes that code for poly(ADP-ribose) polymerase (PARP): parp1 and parp2. Both PARPs are activated by DNA damage caused by, example reactive oxygen species. Upon activation polymers of ADPribose are synthesized on a range of nuclear enzymes using NAD + as substrate. Here, we show that in plants stresses such as drought, high light and heat activate PARP causing NAD + breakdown and ATP consumption.When the PARP activity is reduced by means of chemical inhibitors or by gene silencing, cell death is inhibited and plants become tolerant to a broad range of abiotic stresses like high light, drought and heat. Plant lines with low poly(ADP-ribosyl)ation activity maintain under stress conditions their energy homeostasis by reducing NAD + breakdown and consequently energy consumption. The higher energy-use efficiency avoids the need for a too intense mitochondrial respiration and consequently reduces the formation of reactive oxygen species. From these results it can be concluded that breeding or engineering for a high energy-use efficiency under stress conditions is a valuable, but until today nearly unexploited, approach to enhance overall stress tolerance of crops.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA.Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region. In contrast, the STNV-1 and -2 trailers represent half of the genome and share 64% nucleotide sequence conservation (4). STNV RNA is characterized by the absence of both a cap structure at the 5' end and a poly(A) tail at the 3' end (11). This peculiarity has two implications with regard to translation.First, STNV RNA apparently uses a translational initiation mechanism that bypasses the common cap recognition step. This mechanism may be quite different from that used by other uncapped viral RNAs like those of picornaviruses, comoviruses, and potyviruses (3, 27, 28). Picornaviral RNAs have long 5' untranslated sequences ranging from 600 to 1,200 nt, which carry multiple noninitiating AUG codons and which can fold into highly conserved secondary structures involved in translation initiation (reference 27 and references therein). In contrast, the STNV leader sequence is very short, and its predicted hairpin structure is not as extensive and stable (4). Interestingly, translation of STNV RNA in vitro, as of poliovirus, is stimulated by the cap binding factor eukaryotic initiation factor 4F (eIF-4F) (1, 6).Second, the absence of a poly(A) tail poses the question of how STNV RNA substitutes for the major cytoplasmic functions associated with this structure, i.e., the control of mRNA stability and the modulation of translation efficiency. The poly(A) tail, complexed with poly(A)-binding protein, is believed to enhance the formation of 80S translational initi-* Corresponding author. ation complexes, presumably through promoting some sort of interaction between 5'-and 3'-proximal elements of the mRNA (20). The exact role of the STNV trailer in viral replication and translation is not known, but its regulatory function is suggested by its sequence conservation, which exceeds that of the coat protein coding region. Phylogenetic comparison between the STNV-1 and STNV-2 trailer sequences revealed the presence of three pseudoknotted structures which are also found in several other nonpolyadenylated plant viral RNAs such as that of tobacco mosaic virus (TMV) (4, 23). In the latter case, it was shown that the pseudoknot domain can ...

SummaryThe RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5Ј cap and a 3Ј poly(A) tail. The STNV trailer contains an autonomous translational enhancer domain (TED) that promotes translation in vitro by more than one order of magnitude when combined with the 5Ј-terminal 173 nt of STNV RNA. We now show that the responsible sequence within the 5Ј region maps to the first 38 nt of the STNV RNA. Mutational analysis indicated that the primary sequence of the STNV 5Ј 38 nt and TED is important for translation stimulation in vitro, but did not reveal a role for the complementarity between the two. Translation of chimeric STNV-cat RNAs in tobacco protoplasts showed that TED promotes translation in vivo of RNAs lacking a cap and/or a poly(A) tail. Similar to in vitro, TED-dependent translation in tobacco was stimulated further by the STNV 5Ј 38 nt.

SummaryIn an attempt to understand the feasibility of future targeted genome optimization in agronomic crops, we tested the efficiency of homologous recombination-mediated sequence insertion upon induction of a targeted DNA double-strand break at the desired integration site in maize. By the development of an efficient tissue culture protocol, and with the use of an I-Sce I gene optimized for expression in maize, large numbers of precisely engineered maize events were produced in which DNA integration occurred very accurately.In a subset of events examined in detail, no additional deletions and/or insertions of short filler DNA at the integration site were observed. In 30%-40% of the recovered events, no traces of random insertions were observed. This was true for DNA delivery by both Agrobacterium and particle bombardment. These data suggest that targeted double-strand break-induced homologous recombination is a superior method to generate specific desired changes in the maize genome, and suggest targeted genome optimization of agronomic crops to be feasible.

SummaryIn monocots, transgene expression can be stimulated by over two magnitudes by including an intron in the 5' untranslated region (UTR). The underlying mechanism is presently unknown. Inclusion of the salT intron into the 5' UTR of cat and bar genes stimulated expression of the first gene only, indicating that intron-mediated enhancement of expression (IME) is gene-dependent. Stimulation was associated with increased cat RNA levels, which did not result from a reduced cytoplasmic turnover and were not associated with increased translation. This implies that IME acts in the nucleus. Importantly, the cytoplasmic accumulation of spliced cat transcripts, even with IME, is less than that encoded by the intronless bar gene. As the cat and bar genes were flanked by identical regulatory signals, and the transcripts had a similar cytoplasmic stability, it may mean that IME rescues rather than stimulates gene expression.