Several translation initiation factors in mammals and yeast are regulated by phosphorylation. The phosphorylation state of these factors is subject to alteration during development, environmental stress (heat shock, starvation, or heme deprivation), or viral infection. The phosphorylation state and the effect of changes in phosphorylation of the translation initiation factors of higher plants have not been previously investigated. We have determined the isoelectric states for the wheat translation initiation factors eIF-4A, eIF-4B, eIF-4F, eIFiso4F, and eIF-2 and the poly(A)-binding protein in the seed, during germination, and following heat shock of wheat seedlings using two-dimensional gel electrophoresis and Western analysis. We found that the developmentally induced changes in isoelectric state observed during germination or the stress-induced changes were consistent with changes in phosphorylation. Treatment of the phosphorylated forms of the factors with phosphatases confirmed that the nature of the modification was due to phosphorylation. The isoelectric states of eIF-4B, eIF-4F (eIF-4E, p26), eIF-iso4F (eIFiso4E, p28), and eIF-2␣ (p42) were altered during germination, suggesting that phosphorylation of these factors is developmentally regulated and correlates with the resumption of protein synthesis that occurs during germination. The phosphorylation of eIF-2␤ (p38) or poly(A)-binding protein did not change either during germination or following a thermal stress. Only the phosphorylation state of two factors, eIF-4A and eIF-4B, changed following a heat shock, suggesting that plants may differ significantly from animals in the way in which their translational machinery is modified in response to a thermal stress.Exposure to heat shock results in profound changes at almost every level of gene expression including transcription, splicing, nucleocytoplasmic transport, translation, and protein turnover (for reviews, see Refs. 1-3). Although many of the molecular events involved in heat shock gene induction are remarkably conserved in eukaryotes, the control of translation following heat shock varies considerably among species. In yeast, the heat shock response appears to involve only transcriptional mechanisms, whereas in Xenopus oocytes, the heat shock response is mediated entirely at the translational level (4, 5). As with mammals, the heat shock response in plants lies between these extremes, with gene regulation involving both transcriptional and translational mechanisms. In addition to a specific set of heat-induced genes, i.e. those encoding the heat shock proteins, a low level of non-heat shock mRNA translation continues in plants following heat shock (6 -9). We have shown that heat shock causes a reduction in translational efficiency and an increase in the mRNA half-life of non-heat shock mRNAs in plants that are proportional to the severity of the stress (10). Under these conditions, the translational machinery loses its ability to discriminate between capped and uncapped mRNAs. As a consequence, tra...

Arabidopsis thaliana knockout lines for the plant-specific eukaryotic translation initiation factors eIFiso4G1 (i4g1) and eIFiso4G2 (i4g2) genes have been obtained. To address the potential for functional redundancy of these genes, homozygous double mutant lines were generated by crossing individual knockout lines. Both single and double mutant plants were analyzed for changes in gross morphology, development, and responses to selected environmental stressors. Single gene knockouts appear to have minimal effect on morphology, germination rate, growth rate, flowering time, or fertility. However, double mutant i4g1/i4g2 knockout plants show reduced germination rates, slow growth rates, moderate chlorosis, impaired fertility and reduced long term seed viability. Double mutant plants also exhibit altered responses to dehydration, salinity, and heat stress. The i4g2 and i4g1/i4g2 double mutant has reduced amounts of chlorophyll a and b suggesting a role in the expression of chloroplast proteins. General protein synthesis did not appear to be affected as the levels of gross protein expression did not appear to change in the mutants. The lack of a phenotype for either of the single mutants suggests there is considerable functional overlap. However, the strong phenotypes observed for the double mutant indicates that the individual gene products may have specialized roles in the expression of proteins involved in plant growth and development.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-010-9670-z) contains supplementary material, which is available to authorized users.

Ethylene regulates plant growth in response to many adverse environmental conditions, including the induction of aerenchyma, i.e. the formation of air spaces, in flooded roots in an effort to maintain oxygen levels. In this work, quantitative RT-PCR and in situ RNA hybridization were used to determine how the expression of the ethylene biosynthetic machinery in maize roots is spatially and temporally regulated following exposure to 4% oxygen (i.e. hypoxia) for up to 24 h, conditions that induced aerenchyma formation in the fully-expanded region of the root and reduced cytoplasmic density throughout the root. Expression of ACC oxidase, the ethylene forming enzyme, was observed in the root cap, protophloem sieve elements, and companion cells associated with metaphloem sieve elements. Exposure to 4% oxygen induced ACC oxidase expression in these cell types as well as in the root cortex. ACC synthase, which generates the ethylene precursor, was expressed in the root cap and the cortex and its expression was induced in cortical cells following low oxygen treatment. The induction of expression of the ethylene biosynthetic machinery was accompanied by an induction of ethylene evolution and a reduced rate of root growth. These results suggest that maize roots respond to conditions of hypoxia by inducing the spatially restricted expression of the ethylene biosynthetic machinery, resulting in increased ethylene production.

The plant heat stress protein, Hsp101, and the yeast ortholog, Hsp104, are required to confer thermotolerance in plants and yeast (Saccharomyces cerevisiae), respectively. In addition to its function during stress, Hsp101 is developmentally regulated in plants although its function during development is not known. To determine how the expression of Hsp101 is regulated in cereals, we investigated the Hsp101 expression profile in developing maize (Zea mays). Hsp101 protein was most abundant in the developing tassel, ear, silks, endosperm, and embryo. It was less abundant in the vegetative and floral meristematic regions and was present at only a low level in the anthers and tassel at anthesis, mature pollen, roots, and leaves. As expected, heat treatment resulted in an increase in the level of Hsp101 protein in several organs. In expanding foliar leaves, husk leaves, the tassel at the premeiosis stage of development, or pre-anthesis anthers, however, the heat-mediated increase in protein was not accompanied by an equivalent increase in mRNA. In contrast, the level of Hsp101 transcript increased in the tassel at anthesis following a heat stress without an increase in Hsp101 protein. In other organs such as the vegetative and floral meristematic regions, fully expanded foliar leaves, the young ear, and roots, the heat-induced increase in Hsp101 protein was accompanied by a corresponding increase in Hsp101 transcript level. However, anthers at anthesis, mature pollen, developing endosperm, and embryos largely failed to mount a heat stress response at the level of Hsp101 protein or mRNA, indicating that Hsp101 expression is not heat inducible in these organs. In situ RNA localization analysis revealed that Hsp101 mRNA accumulated in the subaleurone and aleurone of developing kernels and was highest in the root cap meristem and quiescent center of heat-stressed roots. These data suggest an organ-specific control of Hsp101 expression during development and following a heat stress through mechanisms that may include posttranscriptional regulation.Aspects of the response to heat stress have been highly conserved from bacteria to humans, including the induction of heat stress protein (Hsp) synthesis. Heat stress results in the production of mis-folded proteins during their synthesis and the denaturation of existing proteins. Prevention of denaturation or refolding of already denatured proteins appears to be the principle function of the Hsps. Several classes of Hsps have been described in plants including Hsp100, Hsp90, Hsp70, Hsp60, and the small Hsps (for review, see Vierling, 1991; Winter and Sinibaldi, 1991; Miernyk, 1999). The chaperone function of some Hsps, such as Hsp100, has been reported to promote protein disaggregation following a thermal stress (Parsell et al., 1994; Glover and Lindquist, 1998) whereas that of others, such as Hsp70, promotes refolding of denatured proteins once released from the protein aggregates (for review, see Parsell and Lindquist, 1993; Miernyk, 1999).In addition to their function du...

and lnstitute of Mutagenesis and Differentiation, Consiglio Nazionale delle Richerche, via Svezia 1 O, 561 24 Pisa, ltaly (L.P.)l h e effect of heat shock on translational efficiency and message stability of a reporter mRNA was examined in carrot (Daucus carofa). Heat shock of short duration resulted in an increase in protein yield, whereas repression was observed following extended exposure t o the stress. Regardless of the duration of the heat shock, a loss in the function of the 5' cap [m7G(5')ppp(5')N, where N represents any nucleotide] and the 3' poly(A) tail, two regulatory elements that work in concert to establish an efficient leve1 of translation, was observed. This apparent paradox was resolved upon examination of the mRNA half-life following thermal stress, i n which increases up to 1 O-fold were observed. Message stability increased as a function of the severity of the heat shock so that following a mild to moderate stress the increase in message stability more than compensated for the reduction in cap and poly(A) tail function. Following a severe heat shock, the increased mRNA halflife was not sufficient t o overcome the virtual loss in cap and poly(A) tail function. No stimulation of protein synthesis was observed following a heat shock in Chinese hamster ovary cells, data suggesting that the heat-induced increases in mRNA stability may be unique to the heat-shock response i n plants.