No abstract

Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

SummaryGynoecia of the Arabidopsis mutant sty1-1 display abnormal style morphology and altered vascular patterning. These phenotypes, which are enhanced in the sty1-1 sty2-1 double mutant, suggest that auxin homeostasis or signalling might be affected by mutations in STY1 and STY2, both members of the SHI gene family. Chemical inhibition of polar auxin transport (PAT) severely affects the apical-basal patterning of the gynoecium, as do mutations in the auxin transport/signalling genes PIN1, PID and ETT. Here we show that the apical-basal patterning of sty1-1 and sty1-1 sty2-1 gynoecia is hypersensitive to reductions in PAT, and that sty1-1 enhances the PAT inhibition-like phenotypes of pin1-5, pid-8 and ett-1 gynoecia. Furthermore, we show that STY1 activates transcription of the flavin monooxygenase-encoding gene THREAD/YUCCA4, involved in auxin biosynthesis, and that changes in expression of STY1 and related genes lead to altered auxin homeostasis. Our results suggest that STY1 and related genes promote normal development of the style and affect apical-basal patterning of the gynoecium through regulation of auxin homeostasis.

SummaryGene duplication events, and the subsequent functional divergence of duplicates, are believed to be important evolutionary agents, driving morphological diversification. We have studied the structural and functional diversification of members of a plant-specific gene family in Arabidopsis thaliana by analysing mutant phenotypes, expression patterns and phylogeny. The SHI gene family comprises ten members that encode proteins with a RING finger-like zinc finger motif. We show that, despite being highly divergent in sequence, except in two conserved regions, many of the SHI-related genes are partially redundant in function and synergistically promote gynoecium, stamen and leaf development in Arabidopsis. Gynoecia of the loss-offunction sty1-1 mutant display subtle morphological defects, and, although mutations in the related STY2, SHI, SRS3, SRS4, SRS5, SRS7 and LRP1 genes have no apparent effect on gynoecium development, the sty1-1 mutant phenotype is gradually enhanced in double, triple, quadruple and quintuple mutant combinations, suggesting a remarkably extensive functional conservation within the family, which appears to be based on dosage dependency and protection against dominant negative mutations. In multiple mutant lines, all marginal tissues in the apical part of the gynoecium are dramatically reduced or missing, and our data indicate that SHI family members may promote formation of these tissues downstream of the transcriptional corepressor LEUNIG (LUG).

The establishment and maintenance of auxin maxima in vascular plants is regulated by auxin biosynthesis and polar intercellular auxin flow. The disruption of normal auxin biosynthesis in mouse-ear cress (Arabidopsis thaliana) leads to severe abnormalities, suggesting that spatiotemporal regulation of auxin biosynthesis is fundamental for normal growth and development. We have shown previously that the induction of the SHORT-INTERNODES/STYLISH (SHI/STY) family member STY1 results in increased transcript levels of the YUCCA (YUC) family member YUC4 and also higher auxin levels and auxin biosynthesis rates in Arabidopsis seedlings. We have also shown previously that SHI/STY family members redundantly affect development of flowers and leaves. Here, we further examine the function of STY1 by analyzing its DNA and protein binding properties. Our results suggest that STY1, and most likely other SHI/STY members, are DNA binding transcriptional activators that target genes encoding proteins mediating auxin biosynthesis. This suggests that the SHI/STY family members are essential regulators of auxin-mediated leaf and flower development. Furthermore, the lack of a shoot apical meristem in seedlings carrying a fusion construct between STY1 and a repressor domain, SRDX, suggests that STY1, and other SHI/STY members, has a role in the formation and/or maintenance of the shoot apical meristem, possibly by regulating auxin levels in the embryo.

The emergence and radiation of multicellular land plants was driven by crucial innovations to their body plans. The directional transport of the phytohormone auxin represents a key, plant-specific mechanism for polarization and patterning in complex seed plants. Here, we show that already in the early diverging land plant lineage, as exemplified by the moss Physcomitrella patens, auxin transport by PIN transporters is operational and diversified into ER-localized and plasma membrane-localized PIN proteins. Gain-of-function and loss-of-function analyses revealed that PIN-dependent intercellular auxin transport in Physcomitrella mediates crucial developmental transitions in tip-growing filaments and waves of polarization and differentiation in leaf-like structures. Plasma membrane PIN proteins localize in a polar manner to the tips of moss filaments, revealing an unexpected relation between polarization mechanisms in moss tip-growing cells and multicellular tissues of seed plants. Our results trace the origins of polarization and auxin-mediated patterning mechanisms and highlight the crucial role of polarized auxin transport during the evolution of multicellular land plants.

The albino3 (alb3) mutant of Arabidopsis forms white or light yellow cotyledons and leaves and when germinated on soil does not survive beyond the seedling stage. The chloroplasts of the mutant are abnormal, as determined by electron microscopy, and contain reduced levels of chlorophyll. However, the chloroplasts of alb3 mutants are sufficiently differentiated to enable the expression of two nuclear genes whose transcription requires the presence of chloroplasts. The ALB3 gene was isolated by transposon tagging with the Activator/Dissociation transposable element system. ALB3 is a novel plant gene whose product shows homology to a bacterial membrane protein previously identified in five bacterial species and to a yeast protein, OXA1, and its human homolog. OXA1 is required in the mitochondria for proper assembly of the cytochrome oxidase complex. ALB3 does not have a function identical to OXA1 because mitochondrial cytochrome oxidase activity is not affected in the mutant, and immunogold labeling as well as chloroplast import experiments performed in vitro demonstrated that the ALB3 protein is present in chloroplast membranes. ALB3 might have a function related to that of OXA1 and be involved in the assembly of a chloroplast enzyme complex.

In order to establish a reference for analysis of the function of auxin and the auxin biosynthesis regulators SHORT INTERNODE/ STYLISH (SHI/STY) during Physcomitrella patens reproductive development, we have described male (antheridial) and female (archegonial) development in detail, including temporal and positional information of organ initiation. This has allowed us to define discrete stages of organ morphogenesis and to show that reproductive organ development in P. patens is highly organized and that organ phyllotaxis differs between vegetative and reproductive development. Using the PpSHI1 and PpSHI2 reporter and knockout lines, the auxin reporters GmGH3 pro :GUS and PpPINA pro :GFP-GUS, and the auxin-conjugating transgene PpSHI2 pro :IAAL, we could show that the PpSHI genes, and by inference also auxin, play important roles for reproductive organ development in moss. The PpSHI genes are required for the apical opening of the reproductive organs, the final differentiation of the egg cell, and the progression of canal cells into a cell death program. The apical cells of the archegonium, the canal cells, and the egg cell are also sites of auxin responsiveness and are affected by reduced levels of active auxin, suggesting that auxin mediates PpSHI function in the reproductive organs.