Steffen Greiner

Ectopic expression of a tobacco invertase inhibitor homolog prevents cold-induced sweetening of potato tubers.

We have transformed potato with Nt-inhh cDNA, encoding a putative vacuolar homolog of a tobacco cell wall invertase inhibitor, under the control of the CaMV 35S promoter. In transgenic tubers, cold-induced hexose accumulation was reduced by up to 75%, without any effect on potato tuber yield. Processing quality of tubers was greatly improved without changing starch quantity or quality, an important prerequisite for the biotechnological use of Nt-inhh for potato transformation.

Plant Cell Wall Homeostasis Is Mediated by Brassinosteroid Feedback Signaling

Brassinosteroid (BR) signaling is required for normal plant growth as shown by the dwarf phenotype of loss-of-function BR biosynthetic or perception mutants. Despite a detailed understanding of the BR signaling network, it is not clear how exactly BRs control growth. For instance, genetic sector analysis shows that BRs, in contrast to most other growth regulators, act locally, presumably in an autocrine and/or paracrine mode, suggesting that they have some role in feedback regulation. Here, we show that at least one role for BRs in growth control is to ensure pectin-dependent cell wall homeostasis. Pectins are complex block cell wall polymers, which can be modified in the wall by the enzyme pectin methylesterase (PME). Genetic or pharmacological interference with PME activity causes dramatic changes in growth behavior, which are primarily the result of the activation of the BR signaling pathway. We propose that this activation of BR signaling is part of a compensatory response, which protects the plant against the loss of cell wall integrity caused by the imbalance in pectin modification. Thus, feedback signaling from the cell wall is integrated by the BR signaling module to ensure homeostasis of cell wall biosynthesis and remodeling.

Structural Insights into the Target Specificity of Plant Invertase and Pectin Methylesterase Inhibitory Proteins

Pectin methylesterase (PME) and invertase are key enzymes in plant carbohydrate metabolism. Inhibitors of both enzymes constitute a sequence family of extracellular proteins. Members of this family are selectively targeted toward either PME or invertase. In a comparative structural approach we have studied how this target specificity is implemented on homologous sequences. By extending crystallographic work on the invertase inhibitor Nt-CIF to a pectin methylesterase inhibitor (PMEI) from Arabidopsis thaliana, we show an α-helical hairpin motif to be an independent and mobile structural entity in PMEI. Removal of this hairpin fully inactivates the inhibitor. A chimera composed of the α-hairpin of PMEI and the four-helix bundle of Nt-CIF is still active against PME. By contrast, combining the corresponding segment of Nt-CIF with the four-helix bundle of PMEI renders the protein inactive toward either PME or invertase. Our experiments provide insight in how these homologous inhibitors can make differential use of similar structural modules to achieve distinct functions. Integrating our results with previous findings, we present a model for the PME-PMEI complex with important implications.

Growth control by cell wall pectins

Plant cell growth is controlled by the balance between turgor pressure and the extensibility of the cell wall. Several distinct classes of wall polysaccharides and their interactions contribute to the architecture and the emergent features of the wall. As a result, remarkable tensile strength is achieved without relinquishing extensibility. The control of growth and development does not only require a precisely regulated biosynthesis of cell wall components, but also constant remodeling and modification after deposition of the polymers. This is especially evident given the fact that wall deposition and cell expansion are largely uncoupled. Pectins form a functionally and structurally diverse class of galacturonic acid-rich polysaccharides which can undergo abundant modification with a concomitant change in physicochemical properties. This review focuses on homogalacturonan demethylesterification catalyzed by the ubiquitous enzyme pectin methylesterase (PME) as a growth control module. Special attention is drawn to the recently discovered role of this process in primordial development in the shoot apical meristem.

The N‐terminal pro region mediates retention of unprocessed type‐I PME in the Golgi apparatus

The pectin matrix of the cell wall, a complex and dynamic network, impacts on cell growth, cell shape and signaling processes. A hallmark of pectin structure is the methylesterification status of its major component, homogalacturonan (HGA), which affects the biophysical properties and enzymatic turnover of pectin. The pectin methylesterases (PMEs), responsible for de-esterification, encompass a protein family of more than 60 isoforms in the Arabidopsis genome. The pivotal role of PME in the regulation of pectin properties also requires tight control at the post-translational level. Type-I PMEs are characterized by an N-terminal pro region, which exhibits homology with pectin methylesterase inhibitors (PMEIs). Here, we demonstrate that the proteolytic removal of the N-terminal pro region depends on conserved basic tetrad motifs, occurs in the early secretory pathway, and is required for the subsequent export of the PME core domain to the cell wall. In addition, we demonstrate the involvement of AtS1P, a subtilisin-like protease, in Arabidopsis PME processing. Our results indicate that the pro region operates as an effective retention mechanism, keeping unprocessed PME in the Golgi apparatus. Consequently, pro-protein processing could constitute a post-translational mechanism regulating PME activity.

Identification of pollen‐expressed pectin methylesterase inhibitors in Arabidopsis

Pectin methylesterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell wall. Previous work indicated that plant PMEs may be subject to post‐translational regulation. Here, we report the analysis of two proteinaceous inhibitors of PME in Arabidopsis thaliana (AtPMEI1 and 2). The functional analysis of recombinant AtPMEI1 and 2 proteins revealed that both proteins are able to inhibit PME activity from flowers and siliques. Quantitative RT‐PCR analysis indicated that expression of AtPMEI1 and 2 mRNAs is tightly regulated during plant development with highest mRNA levels in flowers. Promotor::GUS fusions demonstrated that expression is mostly restricted to pollen.

In seedlings of the heavy metal accumulator Brassica juncea Cu2+ differentially affects transcript amounts for γ-glutamylcysteine synthetase (γ-ECS) and metallothionein (MT2)

© 1997 Federation of European Biochemical Societies.

A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling

Significance Plant growth and development depend on the biosynthesis and remodeling of the cell wall. To coordinate these two processes, surveillance mechanisms have evolved to monitor the state of the cell wall. The brassinosteroid (BR) hormone signaling pathway plays an essential role in growth control and regulates the expression of a plethora of cell wall-related genes. We have previously shown that feedback signaling from the wall can modulate the outputs of the BR pathway, ensuring cell wall homeostasis and integrity. Here, we identified a receptor-like protein (RLP44), which mediates the activation of BR signaling through direct interaction with the BR coreceptor BAK1. Thus, RLP44 integrates cell wall surveillance with hormone signaling to control cell wall integrity and growth. The brassinosteroid (BR) signaling module is a central regulator of plant morphogenesis, as indicated by the large number of BR-responsive cell wall-related genes and the severe growth defects of BR mutants. Despite a detailed knowledge of the signaling components, the logic of this auto-/paracrine signaling module in growth control remains poorly understood. Recently, extensive cross-talk with other signaling pathways has been shown, suggesting that the outputs of BR signaling, such as gene-expression changes, are subject to complex control mechanisms. We previously provided evidence for a role of BR signaling in a feedback loop controlling the integrity of the cell wall. Here, we identify the first dedicated component of this feedback loop: a receptor-like protein (RLP44), which is essential for the compensatory triggering of BR signaling upon inhibition of pectin de-methylesterification in the cell wall. RLP44 is required for normal growth and stress responses and connects with the BR signaling pathway, presumably through a direct interaction with the regulatory receptor-like kinase BAK1. These findings corroborate a role for BR in controlling the sensitivity of a feedback signaling module involved in maintaining the physico-chemical homeostasis of the cell wall during cell expansion.

In Arabidopsis thaliana, the invertase inhibitors AtC/VIF1 and 2 exhibit distinct target enzyme specificities and expression profiles

Plant cell wall (CWI) and vacuolar invertases (VI) play important roles in carbohydrate metabolism, stress responses and sugar signaling. Addressing the regulation of invertase activities by inhibitor proteins (C/VIF, ell wall/ acuolar nhibitor of ructosidase), we have identified two C/VIFs from Arabidopsis thaliana. AtC/VIF1 showed specific inhibition of VI activity, whereas AtC/VIF2 inhibited both, CWI and VI. Expression analysis revealed that expression of AtC/VIF1 was restricted to specific organs, AtC/VIF2, however, was weakly expressed throughout plant development. Promoter::GUS transformants confirmed pronounced differences of tissue/cell type‐specific expression between both isoforms. Growth of an AtC/VIF1 T‐DNA KO mutant was unaffected, but VI activity and hexose content were slightly increased.

Extracellular invertase is involved in the regulation of clubroot disease in Arabidopsis thaliana.

Clubroot disease of Brassicaceae is caused by an obligate biotrophic protist, Plasmodiophora brassicae. During root gall development, a strong sink for assimilates is developed. Among other genes involved in sucrose and starch synthesis and degradation, the increased expression of invertases has been observed in a microarray experiment, and invertase and invertase inhibitor expression was confirmed using promoter::GUS lines of Arabidopsis thaliana. A functional approach demonstrates that invertases are important for gall development. Different transgenic lines expressing an invertase inhibitor under the control of two root-specific promoters, Pyk10 and CrypticT80, which results in the reduction of invertase activity, showed clearly reduced clubroot symptoms in root tissue with highest promoter expression, whereas hypocotyl galls developed normally. These results present the first evidence that invertases are important factors during gall development, most probably in supplying sugars to the pathogen. In addition, root-specific repression of invertase activity could be used as a tool to reduce clubroot symptoms.