The Plant Journal

Fang, Jun; Chai, Chenglin; Qian, Qian; Li, Chunlai; Tang, Jiuyou; Sun, Lei; Huang, Zejun; Guo, Xiaoli; Sun, Changhui; Liu, Min; Zhang, Yan; Lu, Qingtao; Wang, Yiqin; Lu, Congming; Han, Bin; Chen, Fan; Cheng, Zhukuan; Chu, Chengcai

doi: 10.1111/j.1365-313X.2008.03411.xpmid: 18208525

Pre‐harvest sprouting (PHS) or vivipary in cereals is an important agronomic trait that results in significant economic loss. A considerable number of mutations that cause PHS have been identified in several species. However, relatively few viviparous mutants in rice (Oryza sativa L.) have been reported. To explore the mechanism of PHS in rice, we carried out an extensive genetic screening and identified 12 PHS mutants (phs). Based on their phenotypes, these phs mutants were classified into three groups. Here we characterize in detail one of these groups, which contains mutations in genes encoding major enzymes of the carotenoid biosynthesis pathway, including phytoene desaturase (OsPDS), ζ‐carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene β‐cyclase (β‐OsLCY), which are essential for the biosynthesis of carotenoid precursors of ABA. As expected, the amount of ABA was reduced in all four phs mutants compared with that in the wild type. Chlorophyll fluorescence analysis revealed the occurrence of photoinhibition in the photosystem and decreased capacity for eliminating excess energy by thermal dissipation. The greatly increased activities of reactive oxygen species (ROS) scavenging enzymes, and reduced photosystem (PS) II core proteins CP43, CP47 and D1 in leaves of the Oscrtiso/phs3‐1mutant and OsLCY RNAi transgenic rice indicated that photo‐oxidative damage occurred in PS II, consistent with the accumulation of ROS in these plants. These results suggest that the impairment of carotenoid biosynthesis causes photo‐oxidation and ABA‐deficiency phenotypes, of which the latter is a major factor controlling the PHS trait in rice.

Woo, Mi‐Ok; Ham, Tae‐Ho; Ji, Hyeon‐So; Choi, Min‐Seon; Jiang, Wenzhu; Chu, Sang‐Ho; Piao, Rihua; Chin, Joong‐Hyoun; Kim, Jung‐A; Park, Bong Soo; Seo, Hak Soo; Jwa, Nam‐Soo; McCouch, Susan; Koh, Hee‐Jong

Chang, Chiung‐swey Joanne; Li, Yi‐Hang; Chen, Li‐Teh; Chen, Wan‐Chieh; Hsieh, Wen‐Ping; Shin, Jieun; Jane, Wann‐Neng; Chou, Shu‐Jen; Choi, Giltsu; Hu, Jer‐Ming; Somerville, Shauna; Wu, Shu‐Hsing

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Kitsios, Georgios; Alexiou, Konstantinos G.; Bush, Max; Shaw, Peter; Doonan, John H.

doi: 10.1111/j.1365-313X.2008.03414.xpmid: 18208522

Cyclin‐dependent kinases (CDKs) play key regulatory roles in diverse cellular functions, including cell‐cycle progression, transcription and translation. In plants, CDKs have been classified into several groups, named A through to G, but the functions of most are poorly characterized. CDKCs are known to phosphorylate the C‐terminal domain (CTD) of RNA polymerase II (RNAP II), and therefore the CDKC‐cyclinT (CycT) complex may have a role similar to the animal CDK9‐CycT complex of the positive transcription elongation factor b (P‐TEFb). However, we found that the predicted structure of the Arabidopsis CDKC2 protein is more similar to the mammalian cdc2‐related kinase, CRK7, than to CDK9. CRK7 is proposed to link transcription with splicing, and CDKC2 contains all the structural features of CRK7 that make the latter distinct from CDK9. Consistent with this, we show that GFP‐CDKC2 fusion proteins co‐localize with spliceosomal components, that the expression of CDKC2 modifies the location of these components, and that co‐localization was dependent on the transcriptional status of the cells and on CDKC2‐kinase activity. We propose, therefore, that the Arabidopsis CDKC2 combines the functions of both CRK7 and CDK9, and could also couple splicing with transcription.

Przybyla, Dominika; Göbel, Cornelia; Imboden, André; Hamberg, Mats; Feussner, Ivo; Apel, Klaus

doi: 10.1111/j.1365-313X.2008.03409.xpmid: 18182022

The conditional flu mutant of Arabidopsis accumulates excess amounts of protochlorophyllide within plastid membranes in the dark and generates singlet oxygen upon light exposure. By varying the length of the dark period, the level of the photosensitizer protochlorophyllide may be modulated, and conditions have been established that either endorse the cytotoxicity of 1O2 or reveal its signaling role. Two criteria have been used to distinguish between these two modes of activity of 1O2: the impact of the EXECUTER1 mutation and the prevalence of either non‐enzymatic or enzymatic lipid peroxidation. During illumination of etiolated flu seedlings, toxic effects of 1O2 prevail and non‐enzymatic lipid peroxidation proceeds rapidly. In contrast, in light‐grown flu plants that were subjected to an 8 h dark/light shift, lipid peroxidation occurred almost exclusively enzymatically. The resulting oxidation product, 13‐hydroperoxy octadecatrienoic acid (13‐HPOT), serves as a substrate for synthesis of 12‐oxo phytodienoic acid (OPDA) and jasmonic acid (JA), both of which are known to control various metabolic and developmental processes in plants. Inactivation of the EXECUTER1 protein abrogates not only 1O2‐mediated cell death and growth inhibition of flu plants, but also enzymatic lipid peroxidation. However, inactivation of jasmonate biosynthesis in the aos/flu double mutant does not affect 1O2‐mediated growth inhibition and cell death. Hence, JA and OPDA do not act as second messengers during 1O2 signaling, but form an integral part of a stress‐related signaling cascade activated by 1O2 that encompasses several signaling pathways known to be activated by abiotic and biotic stressors.

Mendoza‐Cózatl, David G.; Butko, Emerald; Springer, Franziska; Torpey, Justin W.; Komives, Elizabeth A.; Kehr, Julia; Schroeder, Julian I.

doi: 10.1111/j.1365-313X.2008.03410.xpmid: 18208526

Phytochelatins (PCs) are glutathione‐derived peptides that function in heavy metal detoxification in plants and certain fungi. Recent research in Arabidopsis has shown that PCs undergo long‐distance transport between roots and shoots. However, it remains unknown which tissues or vascular systems, xylem or phloem, mediate PC translocation and whether PC transport contributes to physiologically relevant long‐distance transport of cadmium (Cd) between shoots and roots. To address these questions, xylem and phloem sap were obtained from Brassica napus to quantitatively analyze which thiol species are present in response to Cd exposure. High levels of PCs were identified in the phloem sap within 24 h of Cd exposure using combined mass spectrometry and fluorescence HPLC analyses. Unexpectedly, the concentration of Cd was more than four‐fold higher in phloem sap compared to xylem sap. Cadmium exposure dramatically decreased iron levels in xylem and phloem sap whereas other essential heavy metals such as zinc and manganese remained unchanged. Data suggest that Cd inhibits vascular loading of iron but not nicotianamine. The high ratios [PCs]/[Cd] and [glutathione]/[Cd] in the phloem sap suggest that PCs and glutathione (GSH) can function as long‐distance carriers of Cd. In contrast, only traces of PCs were detected in xylem sap. Our results suggest that, in addition to directional xylem Cd transport, the phloem is a major vascular system for long‐distance source to sink transport of Cd as PC–Cd and glutathione–Cd complexes.

Hacham, Yael; Matityahu, Ifat; Schuster, Gadi; Amir, Rachel

doi: 10.1111/j.1365-313X.2008.03415.xpmid: 18208521

Methionine and threonine are two essential amino acids, the levels of which limit the nutritional quality of plants. Both amino acids diverge from the same branch of the aspartate family biosynthesis pathway; therefore, their biosynthesis pathways compete for the same carbon/amino substrate. To further elucidate the regulation of methionine biosynthesis and seek ways of increasing the levels of these two amino acids, we crossed transgenic tobacco plants overexpressing the bacterial feedback‐insensitive aspartate kinase (bAK), containing a significantly higher threonine level, with plants overexpressing Arabidopsis cystathionine γ‐synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Plants co‐expressing bAK and the full‐length AtCGS (F‐AtCGS) have significantly higher methionine and threonine levels compared with the levels found in wild‐type plants, but the methionine level does not increase beyond that found in plants expressing F‐AtCGS alone. This finding can be explained through the feedback inhibition regulation mediated by the methionine metabolite on the transcript level of AtCGS. To test this assumption, plants expressing bAK were crossed with plants expressing two mutated forms of AtCGS in which the domains responsible for the feedback regulation have been deleted. Indeed, significantly higher methionine contents and its metabolites levels accumulated in the newly produced plants, and the levels of threonine were also significantly higher than in the wild‐type plants. The transcript level of the two mutated forms of AtCGS significantly increased when there was a high content of threonine in the plants, suggesting that threonine modulates, probably indirectly, the transcript level of AtCGS.

Kim, Hyun Uk; Oostende, Chloë van; Basset, Gilles J.C.; Browse, John

doi: 10.1111/j.1365-313X.2008.03416.xpmid: 18208520

Phylloquinone is the one‐electron carrier at the A1 site of photosystem I, and is essential for photosynthesis. Arabidopsis mutants deficient in early steps of phylloquinone synthesis do not become autotrophic and are seedling lethals, even when grown on sucrose‐supplemented media. Here, we identify acyl‐activating enzyme 14 (AAE14, At1g30520) as the o‐succinylbenzoyl‐coenzyme A (OSB‐CoA) ligase acting in phylloquinone synthesis. Three aae14 mutant alleles, identified by reverse genetics, were found to be seedling lethal, to contain no detectable phylloquinone (< 0.1 pmol mg−1 fresh weight) compared with 10 pmol mg−1 fresh weight in wild‐type leaves, and to accumulate OSB. AAE14 was able to restore menaquinone biosynthesis when expressed in an Escherichia coli mutant disrupted in the menE gene that encodes the bacterial OSB‐CoA ligase. Weak expression of an AAE14 transgene in mutant plants (controlled by the uninduced XVE promoter) resulted in chlorotic, slow‐growing plants that accumulated an average of 4.7 pmol mg−1 fresh weight of phylloquinone. Inducing the XVE promoter in these plants, or expressing an AAE14 transgene under the control of the CaMV 35S promoter, led to full complementation of the mutant phenotype. aae14‐mutant plants were also able to synthesize phylloquinone when provided with 1,4‐dihydroxy‐2‐naphthoate, an intermediate in phylloquinone synthesis downstream of the OSB‐CoA ligase reaction. Expression of an AAE14:GFP reporter construct indicated that the protein accumulated in discrete foci within the chloroplasts. This and other evidence suggests that the enzymes of phylloquinone synthesis from isochorismate may form a complex in the chloroplast stroma to facilitate the efficient channeling of intermediates through the pathway.

Dietrich, Charles R.; Han, Gongshe; Chen, Ming; Berg, R. Howard; Dunn, Teresa M.; Cahoon, Edgar B.

doi: 10.1111/j.1365-313X.2008.03420.xpmid: 18208516

Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and downregulation of this enzyme provides a means for exploring sphingolipid function in cells. We have previously demonstrated that Arabidopsis SPT requires LCB1 and LCB2 subunits for activity, as is the case in other eukaryotes. In this study, we show that Arabidopsis has two genes (AtLCB2a and AtLCB2b) that encode functional isoforms of the LCB2 subunit. No alterations in sphingolipid content or growth were observed in T‐DNA mutants for either gene, but homozygous double mutants were not recoverable, suggesting that these genes are functionally redundant. Reciprocal crosses conducted with Atlcb2a and Atlcb2b mutants indicated that lethality is associated primarily with the inability to transmit the lcb2 null genotype through the haploid pollen. Consistent with this, approximately 50% of the pollen obtained from plants homozygous for a mutation in one gene and heterozygous for a mutation in the second gene arrested during transition from uni‐nucleate microspore to bicellular pollen. Ultrastructural analyses revealed that these pollen grains contained aberrant endomembranes and lacked an intine layer. To examine sphingolipid function in sporophytic cells, Arabidopsis lines were generated that allowed inducible RNAi silencing of AtLCB2b in an Atlcb2a mutant background. Studies conducted with these lines demonstrated that sphingolipids are essential throughout plant development, and that lethality resulting from LCB2 silencing in seedlings could be partially rescued by supplying exogenous long‐chain bases. Overall, these studies provide insights into the genetic and biochemical properties of SPT and sphingolipid function in Arabidopsis.

Vaillant, Isabelle; Tutois, Sylvie; Jasencakova, Zuzana; Douet, Julien; Schubert, Ingo; Tourmente, Sylvette

doi: 10.1111/j.1365-313X.2008.03413.xpmid: 18208523

5S ribosomal DNA (5S rDNA) is organized in tandem repeats on chromosomes 3, 4 and 5 in Arabidopsis thaliana. One part of the 5S rDNA is located within the heterochromatic chromocenters, and the other fraction forms loops with euchromatic features that emanate from the chromocenters. We investigated whether the A. thaliana heterochromatin, and particularly the 5S rDNA, is modified when changing the culture conditions (cultivation in growth chamber versus greenhouse). Nuclei from challenged tissues displayed larger total, as well as 5S rDNA, heterochromatic fractions, and the DNA methyltransferase mutants met1 and cmt3 had different impacts in Arabidopsis. The enlarged fraction of heterochromatic 5S rDNA was observed, together with the reversal of the silencing of some 5S rRNA genes known as minor genes. We observed hypermethylation at CATG sites, and a concomitant DNA hypomethylation at CG/CXG sites in 5S rDNA. Our results show that the asymmetrical hypermethylation is correlated with the ageing of the plants, whereas hypomethylation results from the growth chamber/culture conditions. In spite of severely reduced DNA methylation, the met1 mutant revealed no increase in minor 5S rRNA transcripts in these conditions. The increasing proportion of cytosines in asymmetrical contexts during transition from the euchromatic to the heterochromatic state in the 5S rDNA array suggests that 5S rDNA units are differently affected by the (hypo and hyper)methylation patterns along the 5S rDNA locus. This might explain the different behaviour of 5S rDNA subpopulations inside a 5S array in terms of chromatin compaction and expression, i.e. some 5S rRNA genes would become derepressed, whereas others would join the heterochromatic fraction.