The transient gene expression system using Arabidopsis mesophyll protoplasts has proven an important and versatile tool for conducting cell-based experiments using molecular, cellular, biochemical, genetic, genomic and proteomic approaches to analyze the functions of diverse signaling pathways and cellular machineries. A well-established protocol that has been extensively tested and applied in numerous experiments is presented here. The method includes protoplast isolation, PEG-calcium transfection of plasmid DNA and protoplast culture. Physiological responses and high-throughput capability enable facile and cost-effective explorations as well as hypothesis-driven tests. The protoplast isolation and DNA transfection procedures take 6-8 h, and the results can be obtained in 2-24 h. The cell system offers reliable guidelines for further comprehensive analysis of complex regulatory mechanisms in whole-plant physiology, immunity, growth and development.

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Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.

https://www.cell.com/trends/plant-science/pdf/S1360-1385(09)00298-2.pdf

Proline: a multifunctional amino acid

Proline (Pro) accumulation is a common physiological response in many plants in response to a wide range of biotic and abiotic stresses. Controversy has surrounded the possible role(s) of proline accumulation. In this review, knowledge on the regulation of Pro metabolism during development and stress, results of genetic manipulation of Pro metabolism and current debate on Pro toxicity in plants are presented.

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Proline accumulation in plants: a review.

Plant responses to different stresses are highly complex and involve changes at the transcriptome, cellular, and physiological levels. Recent evidence shows that plants respond to multiple stresses differently from how they do to individual stresses, activating a specific programme of gene expression relating to the exact environmental conditions encountered. Rather than being additive, the presence of an abiotic stress can have the effect of reducing or enhancing susceptibility to a biotic pest or pathogen, and vice versa. This interaction between biotic and abiotic stresses is orchestrated by hormone signalling pathways that may induce or antagonize one another, in particular that of abscisic acid. Specificity in multiple stress responses is further controlled by a range of molecular mechanisms that act together in a complex regulatory network. Transcription factors, kinase cascades, and reactive oxygen species are key components of this cross-talk, as are heat shock factors and small RNAs. This review aims to characterize the interaction between biotic and abiotic stress responses at a molecular level, focusing on regulatory mechanisms important to both pathways. Identifying master regulators that connect both biotic and abiotic stress response pathways is fundamental in providing opportunities for developing broad-spectrum stress-tolerant crop plants.

The interaction of plant biotic and abiotic stresses: from genes to the field

Significance: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. Recent Advances: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria via the electron transport chain. Enhanced ROS production due to proline metabolism has been implicated in the hypersensitive response in plants, lifespan extension in worms, and apoptosis, tumor suppression, and cell survival in animals. Critical Issues: The ability of proline to influence...

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Proline Mechanisms of Stress Survival

SummaryIn vivo protein–protein interactions are frequently studied by means of yeast two-hybrid analysis. However,interactions detected in yeast might differ considerably in the plant system. Based on GAL4 DNA-binding (BD)and activation domains (AD) we established an Arabidopsis protoplast two-hybrid (P2H) system. The use ofGateway -compatible vectors enables the high-throughput screening of protein–protein interactions in plantcells. The efﬁciency of the system was tested by examining the homo- and heterodimerization properties ofbasic leucine zipper (bZIP) transcription factors. A comprehensive heterodimerization matrix of Arabidopsisthaliana group C and group S bZIP transcription factors was generated by comparing the results of yeast andprotoplast two-hybrid experiments. Surprisingly, almost no homodimerization but rather speciﬁc andselective heterodimerization was detected. Heterodimers were preferentially formed between group Cmembers(AtbZIP9,-10,-25,-63) andmembersof groupS1(AtbZIP1,-2,-11,-44,-53). Inaddition,signiﬁcantbutlow-afﬁnity interactions were detected inside group S1, S2 or C AtbZIPs, respectively. As a quantitativeapproach, P2H identiﬁed weak heterodimerization events which were not detected in the yeast system. Thus,in addition to cell biological techniques, P2H is a valuable tool for studying protein–protein interaction in livingplant cells.Keywords: two-hybrid analysis, protein–protein interaction, Arabidopsis thaliana, groups C and S bZIPtranscription factors, heterodimerization.IntroductionProtein–protein interaction has been considered as increas-ingly important for the molecular understanding of biologi-cal processes (Pawson and Nash, 2003). Several methodshavebeenproposedforstudyingprotein–proteininteractionin vitro and in vivo. In particular, yeast two-hybrid approa-ches have been very successful in identifying putative pro-tein interaction partners (Fields and Song, 1989). However,the data obtained from yeast and plant cells might differconsiderably, and therefore, in planta conﬁrmation of theinteraction is necessary. Biochemical approaches like co-immunoprecipitation (Co-IP) or cytological methods likeﬂuorescence resonance energy transfer (FRET) (Wallrabeand Periasamy, 2005) or bimolecular ﬂuorescence comple-mentation (BiFC)(HuandKerppola,2003;Huang et al.,2005;Walter et al., 2004) have been used. The latter are ofadvantage in identifying the cytological localization of agiven protein interaction. However, these methods do notquantify the afﬁnity of the protein partners. Furthermore,

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2006.02731.x

Two‐hybrid protein–protein interaction analysis in Arabidopsis protoplasts: establishment of a heterodimerization map of group C and group S bZIP transcription factors

Proline metabolism has been implicated in plant responses to abiotic stresses. The Arabidopsis thaliana proline dehydrogenase (ProDH) is catalysing the first step in proline degradation. Transcriptional activation of ProDH by hypo-osmolarity is mediated by an ACTCAT cis element, a typical binding site of basic leucine zipper (bZIP) transcription factors. In this study, we demonstrate by gain-of-function and loss-of-function approaches, as well as chromatin immunoprecipitation (ChIP), that ProDH is a direct target gene of the group-S bZIP factor AtbZIP53. Dimerisation studies making use of yeast and Arabidopsis protoplast-based two-hybrid systems, as well as bimolecular fluorescence complementation (BiFC) reveal that AtbZIP53 does not preferentially form dimers with group-S bZIPs but strongly interacts with members of group-C. In particular, a synergistic interplay of AtbZIP53 and group-C AtbZIP10 was demonstrated by colocalisation studies, strong enhancement of ACTCAT-mediated transcription as well as complementation studies in atbzip53 atbzip10 T-DNA insertion lines. Heterodimer mediated activation of transcription has been found to operate independent of the DNA-binding properties and is described as a crucial mechanism to modulate transcription factor activity and function.

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Caroline S. Mayer

Papers

Two‐hybrid protein–protein interaction analysis in Arabidopsis protoplasts: establishment of a heterodimerization map of group C and group S bZIP transcription factors

Combinatorial control of Arabidopsis proline dehydrogenase transcription by specific heterodimerisation of bZIP transcription factors