Boronic acids as tools to study (plant) developmental processes?

Boron (B) is an essential micronutrient for organisms. In plants, B is known to stabilize the cell wall by crosslinking Rhamnogalacturonan II through ester bonds formed with cis-diols of sugar moieties. However, B is believed to be required for additional functions such as stability and function of (plasma membrane) proteins involved in signal transduction pathways. We have recently shown that boronic acids, competitors of B, efficiently induce perfect phenocopies of monopteros mutants. This effect is enigmatic because like B, boronic acids should find numerous cellular targets and thus disturb many biologic processes ending in a spectrum of unspecific embryo phenotypes. Based on chemical characteristics of boronic acids and their derivatives we discuss reasons that could explain this unusual specificity. The peculiarities of this class of compounds could provide new tools for studying developmental processes.

Boronic acid treatment phenocopies monopteros by affecting PIN1 membrane stability and polar auxin transport in Arabidopsis thaliana embryos.

Several observations suggest that the micronutrient boron (B) has a stabilising role in the plasma membrane (PM), supporting functions in PM-linked (hormone) signalling processes. However, this role is poorly characterised. Here we show treatment with boronic acids, specific competitors of B, phenocopies the Arabidopsis thaliana rootless pattern mutant monopteros. At least in part, this is caused by phenylboronic acid (PBA)-induced internalisation of the membrane-localised auxin efflux carrier PINFORMED1 (PIN1) in the early embryo. PIN1 internalisation interrupts the feedback signal transduction cascade involving the phytohormone auxin, PIN1 and the transcription factor gene MONOPTEROS This entails several effects, including abnormal development of vascular cell precursors, suppression of MONOPTEROS downstream targets and loss of the root auxin maximum - essential signals for root meristem development. While PIN1 is internalised, we observe a differential effect of PBA on other proteins, which are either unaffected, internalised or, as in the case of the B transporter BOR1, stabilised at the PM. These findings suggest a competition of PBA with B for plant membrane proteins and might shed light on the function of B at the PM.

Ectopic shoot meristem generation in monocotyledonous rpk1 mutants is linked to SAM loss and altered seedling morphology.

BACKGROUND: In dicot Arabidopsis thaliana embryos two cotyledons develop largely autonomously from the shoot apical meristem (SAM). Recessive mutations in the Arabidopsis receptor-like kinase RPK1 lead to monocotyledonous seedlings, with low (10 %) penetrance due to complex functional redundancy. In strong rpk1 alleles, about 10 % of these (i. e. 1 % of all homozygotes) did not develop a SAM. We wondered whether RPK1 might also control SAM gene expression and SAM generation in addition to its known stochastic impact on cell division and PINFORMED1 (PIN1) polarity in the epidermis. RESULTS: SAM-less seedlings developed a simple morphology with a straight and continuous hypocotyl-cotyledon structure lacking a recognizable epicotyl. According to rpk1's auxin-related PIN1 defect, the seedlings displayed defects in the vascular tissue. Surprisingly, SAM-less seedlings variably expressed essential SAM specific genes along the hypocotyl-cotyledon structure up into the cotyledon lamina. Few were even capable of developing an ectopic shoot meristem (eSM) on top of the cotyledon. CONCLUSIONS: The results highlight the developmental autonomy of the SAM vs. cotyledons and suggest that the primary rpk1 defect does not lie in the seedling's ability to express SAM genes or to develop a shoot meristem. Rather, rpk1's known defects in cell division and auxin homeostasis, by disturbed PIN1 polarity, impact on SAM and organ generation. In early embryo stages this failure generates a simplified monocotyledonous morphology. Once generated, this likely entails a loss of positional information that in turn affects the spatiotemporal development of the SAM. SAM-bearing and SAM-less monocotyledonous phenotypes show morphological similarities either to real monocots or to dicot species, which only develop one cotyledon. The specific cotyledon defect in rpk1 mutants thus sheds light upon the developmental implications of the transition from two cotyledons to one.

Mutations in the Arabidopsis RPK1 gene uncouple cotyledon anlagen and primordia by modulating epidermal cell shape and polarity.

Plant seedlings have either one or two cotyledons. The mechanisms that regulate this organ number are poorly understood. Mutations in the RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) gene of the dicot Arabidopsis have only one cotyledon, with low penetrance due to complex genetic redundancy. An analysis of patterning genes required for cotyledon initiation showed that these have normal expression patterns, defining the cotyledon anlagen, in rpk1. This was also true for key genes, which organize the shoot apical meristem (SAM). By contrast, epidermal cell shape and polarity were compromised in rpk1 embryos, as evidenced by disturbed polarity of the auxin efflux carrier PIN1. PIN1 is required for the establishment of auxin maxima, which induce and maintain organ primordia. The effects in rpk1 mutants manifest in a spatially and timely stochastic fashion probably due to redundancy of RPK1-like functions. Consistently, auxin maxima showed a stochastic distribution in rpk1 embryos, being at times entirely absent and at other times supernumerary. This variability may explain how monocotyledonous seedlings and cotyledon shape variants can developmentally arise in Arabidopsis and possibly in other plants.

Impact of natural genetic variation on the transcriptome of autotetraploid Arabidopsis thaliana.

Polyploidy, the presence of more than two complete sets of chromosomes in an organism, has significantly shaped the genomes of angiosperms during evolution. Two forms of polyploidy are often considered: allopolyploidy, which originates from interspecies hybrids, and autopolyploidy, which originates from intraspecies genome duplication events. Besides affecting genome organization, polyploidy generates other genetic effects. Synthetic allopolyploid plants exhibit considerable transcriptome alterations, part of which are likely caused by the reunion of previously diverged regulatory hierarchies. In contrast, autopolyploids have relatively uniform genomes, suggesting lower alteration of gene expression. To evaluate the impact of intraspecies genome duplication on the transcriptome, we generated a series of unique Arabidopsis thaliana autotetraploids by using different ecotypes. A. thaliana autotetraploids show transcriptome alterations that strongly depend on their parental genome composition and include changed expression of both new genes and gene groups previously described from allopolyploid Arabidopsis. Alterations in gene expression are stable, nonstochastic, developmentally specific, and associated with changes in DNA methylation. We propose that Arabidopsis possesses an inherent and heritable ability to sense and respond to elevated, yet balanced chromosome numbers. The impact of natural variation on alteration of autotetraploid gene expression stresses its potential importance in the evolution and breeding of plants.

The Sos-recruitment system as a tool to analyze cellular localization of plant proteins: membrane localization of Arabidopsis thaliana PEPINO/PASTICCINO2.

We have explored a modified cytosolic yeast-two-hybrid Sos-recruitment system (SRS) in order to test for membrane localization of a protein. In this system, membrane localization is assessed by rescue of a yeast strain carrying a temperature-sensitive mutation in the CDC25 gene (cdc25-2) at restrictive temperature. The homologous human Sos (hSos) is capable to replace cdc25-2 provided that it is attached to the membrane because only then hSos is functional. This can be achieved when hSos is artificially fused to a protein containing trans-membrane domains (Tms). GFP/YFP fusion construct analyses of the Arabidopsis thaliana PEPINO/PASTICCINO2 (PEP/PAS2) protein have previously shown disparate cellular localizations although this protein possesses clear Tms. Analysis of N-terminal and C-terminal hSos-PEP/PAS2 fusions respectively suggests, that PEP/PAS2 is an integral membrane protein with cytosolic N- and C-termini. This implies that the protein has an even number of Tms and that the first Tm, a signal peptide, is not cleaved off. Our study shows that SRS is suitable to test for protein membrane localization and possibly for more detailed topological analysis of membrane proteins.

The gene MACCHI-BOU 4/ENHANCER OF PINOID encodes a NPH3-like protein and reveals similarities between organogenesis and phototropism at the molecular level.

Intercellular transport of the phytohormone auxin is a significant factor for plant organogenesis. To investigate molecular mechanisms by which auxin controls organogenesis, we analyzed the macchi-bou 4 (mab4) mutant identified as an enhancer of pinoid (pid). Although mab4 and pid single mutants displayed relatively mild cotyledon phenotypes, pid mab4 double mutants completely lacked cotyledons. We found that MAB4 was identical to ENHANCER OF PINOID (ENP), which has been suggested to control PIN1 polarity in cotyledon primordia. MAB4/ENP encodes a novel protein, which belongs to the NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) family thought to function as a signal transducer in phototropism and control lateral translocation of auxin. MAB4/ENP mRNA was detected in the protodermal cell layer of the embryo and the meristem L1 layer at the site of organ initiation. In the mab4 embryo, the abundance of PIN1:GFP was severely decreased at the plasma membrane in the protodermal cell layer. In addition, subcellular localization analyses indicated that MAB4/ENP resides on a subpopulation of endosomes as well as on unidentified intracellular compartments. These results indicate that MAB4/ENP is involved in polar auxin transport in organogenesis.

Partial loss-of-function alleles reveal a role for GNOM in auxin transport-related, post-embryonic development of Arabidopsis.

The Arabidopsis GNOM gene encodes an ARF GDP/GTP exchange factor involved in embryonic axis formation and polar localisation of the auxin efflux regulator PIN1. To examine whether GNOM also plays a role in post-embryonic development and to clarify its involvement in auxin transport, we have characterised newly isolated weak gnom alleles as well as trans-heterozygotes of complementing strong alleles. These genotypes form a phenotypic series of GNOM activity in post-embryonic development, with auxin-related defects, especially in the maintenance of primary root meristem activity and in the initiation and organisation of lateral root primordia. Our results suggest a model for GNOM action mediating auxin transport in both embryogenesis and post-embryonic organ development.

The Arabidopsis gene PEPINO/PASTICCINO2 is required for proliferation control of meristematic and non-meristematic cells and encodes a putative anti-phosphatase.

Growth in organisms requires that cell division versus differentiation are precisely controlled and that cells, once differentiated, do not resume proliferation activity. Mutants of the Arabidopsis gene PEPINO/PASTICCINO2 develop abnormal shoot phenotypes from slow tumour-like cell proliferation. Abnormal proliferation is enhanced in a mutant background, which elevates endogenous cytokinin levels as seen in double mutants of pepino and altered meristem primordia1. The enlarged shoot apical meristem produces supernumerary abnormal leaves. However, comparison of expression patterns of Cyclin1At and the homeobox gene KNAT2 indicates that PEPINO ( PEP) is involved in cell cycle regulation of meristematic and non-meristematic cells. PEP encodes a putative anti-phosphatase, a representative of an enigmatic class of proteins, which might participate in proliferation processes. In mammals at least two proteins, Sbf1 and STYX, have been predicted to represent functional anti-phosphatases controlling cell proliferation. However, the in vivo function of this class of molecules is not known since no loss-of-function mutants have been reported to date. Anti-phosphatases display a natural change of the essential cysteine by glycine in the phosphatase catalytic signature motif HCxxGxxR(S/T). The remaining motif harbours highly conserved amino acid residues and is thought to constitute a pocket that enables such proteins to bind but not catalyse phosphorylated residues. One of the sequenced mutations in PEP indicates that the integrity of this anti-phosphatase signature motif is essential for function. The analysis of pepino, the first known loss-of-function mutant of an anti-phosphatase in a multicellular organism, proves the biological significance of these molecules.