Differential responses of anthers of stress tolerant and sensitive wheat cultivars to high temperature stress.

MAIN CONCLUSION: Transcriptomic analyses identified anther-expressed genes in wheat likely to contribute to heat tolerance and hence provide useful genetic markers. The genes included those involved in hormone biosynthesis, signal transduction, the heat shock response and anther development. Pollen development is particularly sensitive to high temperature heat stress. In wheat, heat-tolerant and heat-sensitive cultivars have been identified, although the underlying genetic causes for these differences are largely unknown. The effects of heat stress on the developing anthers of two heat-tolerant and two heat-sensitive wheat cultivars were examined in this study. Heat stress (35 °C) was found to disrupt pollen development in the two heat-sensitive wheat cultivars but had no visible effect on pollen or anther development in the two heat-tolerant cultivars. The sensitive anthers exhibited a range of developmental abnormalities including an increase in unfilled and clumped pollen grains, abnormal pollen walls and a decrease in pollen viability. This subsequently led to a greater reduction in grain yield in the sensitive cultivars following heat stress. Transcriptomic analyses of heat-stressed developing wheat anthers of the four cultivars identified a number of key genes which may contribute to heat stress tolerance during pollen development. Orthologs of some of these genes in Arabidopsis and rice are involved in regulation of the heat stress response and the synthesis of auxin, ethylene and gibberellin. These genes constitute candidate molecular markers for the breeding of heat-tolerant wheat lines.

MYB80 homologues in Arabidopsis, cotton and Brassica: regulation and functional conservation in tapetal and pollen development.

BACKGROUND: The Arabidopsis AtMYB80 transcription factor regulates genes involved in pollen development and controls the timing of tapetal programmed cell death (PCD). Downregulation of AtMYB80 expression precedes tapetal degradation. Inhibition of AtMYB80 expression results in complete male sterility. Full-length AtMYB80 homologs have been isolated in wheat, rice, barley and canola (C genome). RESULTS: The complete sequences of MYB80 genes from the Brassica. napus (A gene), B. juncea (A gene), B. oleracea (C gene) and the two orthologs from cotton (Gossypium hirsutum) were determined. The deduced amino acid sequences possess a highly conserved MYB domain, 44-amino acid region and 18-amino acid C-terminal sequence. The cotton MYB80 protein can fully restore fertility of the atmyb80 mutant, while removal of the 44 amino acid sequence abolishes its function. Two conserved MYB cis-elements in the AtMYB80 promoter are required for downregulation of MYB80 expression in anthers, apparently via negative auto-regulation. In cotton, tapetal degradation occurs at a slightly earlier stage of anther development than in Arabidopsis, consistent with an earlier increase and subsequent downregulation in GhMYB80 expression. The MYB80 homologs fused with the EAR repressor motif have been shown to induce male sterility in Arabidopsis. Constructs were designed to maximize the level of male sterility. CONCLUSIONS: MYB80 genes are conserved in structure and function in all monocot and dicot species so far examined. Expression patterns of MYB80 in these species are also highly similar. The reversible male sterility system developed in Arabidopsis by manipulating MYB80 expression should be applicable to all major crops.

The MYB80 transcription factor is required for pollen development and the regulation of tapetal programmed cell death in Arabidopsis thaliana.

Arabidopsis thaliana MYB80 (formerly MYB103) is expressed in the tapetum and microspores between anther developmental stages 6 and 10. MYB80 encodes a MYB transcription factor that is essential for tapetal and pollen development. Using microarray analysis of anther mRNA, we identified 404 genes differentially expressed in the myb80 mutant. Employing the glucocorticoid receptor system, the expression of 79 genes was changed when MYB80 function was restored in the myb80 mutant following induction by dexamethasone. Thirty-two genes were analyzed using chromatin immunoprecipitation, and three were identified as direct targets of MYB80. The genes encode a glyoxal oxidase (GLOX1), a pectin methylesterase (VANGUARD1), and an A1 aspartic protease (UNDEAD). All three genes are expressed in the tapetum and microspores. Electrophoretic mobility shift assays confirmed that MYB80 binds to all three target promoters, with the preferential binding site containing the CCAACC motif. TUNEL assays showed that when UNDEAD expression was silenced using small interfering RNA, premature tapetal and pollen programmed cell death occurred, resembling the myb80 mutant phenotype. UNDEAD possesses a mitochondrial targeting signal and may hydrolyze an apoptosis-inducing protein(s) in mitochondria. The timing of tapetal programmed cell death is critical for pollen development, and the MYB80/UNDEAD system may regulate that timing.

The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants.

Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate K(m) of 7.5 µm and V(max) of 1800 nmol min(-1) mg(-1) of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg(2+) or Mn(2+). Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains.

Anther Morphological Development and Stage Determination in Triticum aestivum.

Anther development progresses through 15 distinct developmental stages in wheat, and accurate determination of anther developmental stages is essential in anther and pollen studies. A detailed outline of the development of the wheat anther through its entire developmental program, including the 15 distinct morphological stages, is presented. In bread wheat (Triticum aestivum), anther developmental stages were correlated with five measurements, namely auricle distance, spike length, spikelet length, anther length and anther width. Spike length and auricle distance were shown to be suitable for rapid anther staging within cultivars. Anther length is an accurate measurement in determining anther stages and may be applicable for use between cultivars. Tapetal Programmed Cell Death (PCD) in wheat begins between late tetrad stage (stage 8) and the early young microspore stage (stage 9) of anther development. Tapetal PCD continues until the vacuolate pollen stage (stage 11), at which point the tapetum fully degrades. The timing of tapetal PCD initiation is slightly delayed compared to that in rice, but is two stages earlier than in the model dicot Arabidopsis. The MYB80 gene, which encodes a transcription factor regulating the timing of tapetal PCD, reaches its peak expression at the onset of tapetal PCD in wheat.

Suppression and restoration of male fertility using a transcription factor.

The Arabidopsis AtMYB103 gene codes for an R2R3 MYB domain protein whose expression is restricted to the tapetum of developing anthers and to trichomes. Down-regulation of expression using anti-sense leads to abnormal tapetum and pollen development, although seed setting still occurs (Higginson, T., Li, S.F. and Parish, R.W. (2003) AtMYB103 regulates tapetum and trichome development in Arabidopsis thaliana. Plant J. 35, 177-192). In this study, we show that blocking the function of the AtMYB103 gene, employing either an insertion mutant or an AtMYB103EAR chimeric repressor construct under the control of the AtMYB103 promoter, results in complete male sterility and failure to set seed. These plants exhibit similar abnormalities in tapetum and pollen development, with the tapetum becoming highly vacuolated at early stages and degenerating prematurely. No exine is deposited on to the pollen wall. The degeneration of pollen grains commences prior to pollen mitosis, the pollen collapsing and largely lacking cytoplasmic content. A restorer containing the AtMYB103 gene under the control of a stronger anther-specific promoter was introduced into pollen donor plants and crossed into the male sterile plants transgenic for the repressor. The male fertility of F1 plants was restored. The chimeric repressor and the restorer constitute a reversible male sterility system which could be adapted for hybrid seed production. This is the first reversible male sterility system targeting a transcription factor essential for pollen development. Strategies for generating inducible male sterility and maintainable male sterility for the production of hybrid crops are discussed.

The Mycoplasma hyorhinis p37 Protein Rapidly Induces Genes in Fibroblasts Associated with Inflammation and Cancer.

The p37 protein at the surface of Mycoplasma hyorhinis cells forms part of a high-affinity transport system and has been found associated with animal and human cancers. Here we show in NIH3T3 fibroblasts, p37 rapidly induces the expression of genes implicated in inflammation and cancer progression. This gene activation was principally via the Tlr4 receptor. Activity was lost from p37 when the C-terminal 20 amino acids were removed or the four amino acids specific for the hydrogen bonding of thiamine pyrophosphate had been replaced by valine. Blocking the IL6 receptor or inhibiting STAT3 signalling resulted in increased p37-induced gene expression. Since cancer associated fibroblasts support growth, invasion and metastasis via their ability to regulate tumour-related inflammation, the rapid induction in fibroblasts of pro-inflammatory genes by p37 might be expected to influence cancer development.

Tapetal development and abiotic stress: a centre of vulnerability.

Many self-fertilising crops are particularly sensitive to abiotic stress at the reproductive stage. In rice (Oryza sativa L.) and wheat (Triticum aestivum L.), for example, abiotic stress during meiosis and the young microspore stage indicates the tapetum is highly vulnerable and that the developmental program appears to be compromised. Tapetal hypertrophy can occur as a consequence of cold and drought stress, and programmed cell death (PCD) is delayed or inhibited. Since the correct timing of tapetal PCD is essential for pollen reproduction, substantial losses in grain yield occur. In wheat and rice, a decrease in tapetal cell wall invertase levels is correlated with pollen abortion and results in the amount of hexose sugars reaching the tapetum, and subsequently the developing microspores, being severely reduced ('starvation hypothesis'). ABA and gibberellin levels may be modified by cold and drought, influencing levels of cell wall invertase(s) and the tapetal developmental program, respectively. Many genes regulating tapetal and microspore development have been identified in Arabidopsis thaliana (L.) Heynh. and rice and the specific effects of abiotic stresses on the program and pathways can now begin to be assessed.