Maintenance of abiotic stress memory in plants: Lessons learned from heat acclimation.

Plants acquire enhanced tolerance to intermittent abiotic stress by employing information obtained during prior exposure to an environmental disturbance, a process known as acclimation or defense priming. The capacity for stress memory is a critical feature in this process. The number of reports related to plant stress memory (PSM) has recently increased, but few studies have focused on the mechanisms that maintain PSM. Identifying the components involved in maintaining PSM is difficult due in part to the lack of clear criteria to recognize these components. In this review, based on what has been learned from genetic studies on heat acclimation memory, we propose criteria for identifying components of the regulatory networks that maintain PSM. We provide examples of the regulatory circuits formed by effectors and regulators of PSM. We also highlight strategies for assessing PSMs, update the progress in understanding the mechanisms of PSM maintenance, and provide perspectives for the further development of this exciting research field.

Chlorophyll dephytylase 1 and chlorophyll synthase: a chlorophyll salvage pathway for the turnover of photosystems I and II.

Chlorophyll (Chl) is made up of the tetrapyrrole chlorophyllide and phytol, a diterpenoid alcohol. The photosynthetic protein complexes utilize Chl for light harvesting to produce biochemical energy for plant development. However, excess light and adverse environmental conditions facilitate generation of reactive oxygen species, which damage photosystems I and II (PSI and PSII) and induce their turnover. During this process, Chl is released, and is thought to be recycled via dephytylation and rephytylation. We previously demonstrated that Chl recycling in Arabidopsis under heat stress is mediated by the enzymes chlorophyll dephytylase 1 (CLD1) and chlorophyll synthase (CHLG) using chlg and cld1 mutants. Here, we show that the mutants with high CLD1/CHLG ratio, by different combinations of chlg-1 (a knock-down mutant) and the hyperactive cld1-1 alleles, develop necrotic leaves when grown under long- and short-day, but not continuous light conditions, owing to the accumulation of chlorophyllide in the dark. Combination of chlg-1 with cld1-4 (a knock-out mutant) leads to reduced chlorophyllide accumulation and necrosis. The operation of CLD1 and CHLG as a Chl salvage pathway was also explored in the context of Chl recycling during the turnover of Chl-binding proteins of the two photosystems. CLD1 was found to interact with CHLG and the light-harvesting complex-like proteins OHP1 and LIL3, implying that auxiliary factors are required for this process.

SUMOylome Profiling Reveals a Diverse Array of Nuclear Targets Modified by the SUMO Ligase SIZ1 during Heat Stress.

The posttranslational addition of small ubiquitin-like modifier (SUMO) is an essential protein modification in plants that provides protection against numerous environmental challenges. Ligation is accomplished by a small set of SUMO ligases, with the SAP-MIZ domain-containing SIZ1 and METHYL METHANESULFONATE-SENSITIVE21 (MMS21) ligases having critical roles in stress protection and DNA endoreduplication/repair, respectively. To help identify their corresponding targets in Arabidopsis thaliana, we used siz1 and mms21 mutants for proteomic analyses of SUMOylated proteins enriched via an engineered SUMO1 isoform suitable for mass spectrometric studies. Through multiple data sets from seedlings grown at normal temperatures or exposed to heat stress, we identified over 1000 SUMO targets, most of which are nuclear localized. Whereas no targets could be assigned to MMS21, suggesting that it modifies only a few low abundance proteins, numerous targets could be assigned to SIZ1, including major transcription factors, coactivators/repressors, and chromatin modifiers connected to abiotic and biotic stress defense, some of which associate into multisubunit regulatory complexes. SIZ1 itself is also a target, but studies with mutants protected from SUMOylation failed to uncover a regulatory role. The catalog of SIZ1 substrates indicates that SUMOylation by this ligase provides stress protection by modifying a large array of key nuclear regulators.

Distinct heat shock factors and chromatin modifications mediate the organ-autonomous transcriptional memory of heat stress.

Plants can be primed by a stress cue to mount a faster or stronger activation of defense mechanisms upon subsequent stress. A crucial component of such stress priming is the modified reactivation of genes upon recurring stress; however, the underlying mechanisms of this are poorly understood. Here, we report that dozens of Arabidopsis thaliana genes display transcriptional memory, i.e. stronger upregulation after a recurring heat stress, that lasts for at least 3 days. We define a set of transcription factors involved in this memory response and show that the transcriptional memory results in enhanced transcriptional activation within minutes of the onset of a heat stress cue. Further, we show that the transcriptional memory is active in all tissues. It may last for up to a week, and is associated during this time with histone H3 lysine 4 hypermethylation. This transcriptional memory is cis-encoded, as we identify a promoter fragment that confers memory onto a heterologous gene. In summary, heat-induced transcriptional memory is a widespread and sustained response, and our study provides a framework for future mechanistic studies of somatic stress memory in higher plants.

Identification of a Chlorophyll Dephytylase Involved in Chlorophyll Turnover in Arabidopsis.

Chlorophyll turns over in green organs during photosystem repair and is salvaged via de- and rephytylation, but the enzyme involved in dephytylation is unknown. We have identified an Arabidopsis thaliana thylakoid protein with a putative hydrolase domain that can dephytylate chlorophyll in vitro and in vivo. The corresponding locus, CHLOROPHYLL DEPHYTYLASE1 (CLD1), was identified by mapping a semidominant, heat-sensitive, missense allele (cld1-1). CLD1 is conserved in oxygenic photosynthetic organisms, sharing structural similarity with pheophytinase, which functions in chlorophyll breakdown during leaf senescence. Unlike pheophytinase, CLD1 is predominantly expressed in green organs and can dephytylate chlorophyll in vitro. The specific activity is significantly higher for the mutant protein encoded by cld1-1 than the wild-type enzyme, consistent with the semidominant nature of the cld1-1 mutation. Supraoptimal CLD1 activities in cld1-1 mutants and transgenic seedlings led to the proportional accumulation of chlorophyllides derived from chlorophyll dephytylation after heat shock, which resulted in light-dependent cotyledon bleaching. Reducing CLD1 expression diminished thermotolerance and the photochemical efficiency of photosystem II under prolonged moderate heat stress. Taken together, our results suggest that CLD1 is the long-sought enzyme for removing the phytol chain from chlorophyll during its turnover at steady state within the chloroplast.

Heat Shock Protein HSP101 Affects the Release of Ribosomal Protein mRNAs for Recovery after Heat Shock.

Heat shock (HS) is known to have a profound impact on gene expression at different levels, such as inhibition of protein synthesis, in which HS blocks translation initiation and induces the sequestration of mRNAs into stress granules (SGs) or P-bodies for storage and/or decay. SGs prevent the degradation of the stored mRNAs, which can be reengaged into translation in the recovery period. However, little is known on the mRNAs stored during the stress, how these mRNAs are released from SGs afterward, and what the functional importance is of this process. In this work, we report that Arabidopsis HEAT SHOCK PROTEIN101 (HSP101) knockout mutant (hsp101) presented a defect in translation recovery and SG dissociation after HS Using RNA sequencing and RNA immunoprecipitation approaches, we show that mRNAs encoding ribosomal proteins (RPs) were preferentially stored during HS and that these mRNAs were released and translated in an HSP101-dependent manner during recovery. By 15N incorporation and polysome profile analyses, we observed that these released mRNAs contributed to the production of new ribosomes to enhance translation. We propose that, after HS, HSP101 is required for the efficient release of RP mRNAs from SGs resulting in a rapid restoration of the translation machinery by producing new RPs.

Heat-induced ribosome pausing triggers mRNA co-translational decay in Arabidopsis thaliana.

The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5'-ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5'-ribosome pausing leading to the XRN4-mediated 5'-directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20°C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of 'non-aberrant' mRNAs.

Analysis of an Arabidopsis heat-sensitive mutant reveals that chlorophyll synthase is involved in reutilization of chlorophyllide during chlorophyll turnover.

Chlorophylls, the most abundant pigments in the photosynthetic apparatus, are constantly turned over as a result of the degradation and replacement of the damage-prone reaction center D1 protein of photosystem II. Results from isotope labeling experiments suggest that chlorophylls are recycled by reutilization of chlorophyllide and phytol, but the underlying mechanism is unclear. In this study, by characterization of a heat-sensitive Arabidopsis mutant we provide evidence of a salvage pathway for chlorophyllide a. A missense mutation in CHLOROPHYLL SYNTHASE (CHLG) was identified and confirmed to be responsible for a light-dependent, heat-induced cotyledon bleaching phenotype. Following heat treatment, mutant (chlg-1) but not wild-type seedlings accumulated a substantial level of chlorophyllide a, which resulted in a surge of phototoxic singlet oxygen. Immunoblot analysis suggested that the mutation destabilized the chlorophyll synthase proteins and caused a conditional blockage of esterification of chlorophyllide a after heat stress. Accumulation of chlorophyllide a after heat treatment occurred during recovery in the dark in the light-grown but not the etiolated seedlings, suggesting that the accumulated chlorophyllides were not derived from de novo biosynthesis but from de-esterification of the existing chlorophylls. Further analysis of the triple mutant harboring the CHLG mutant allele and null mutations of CHLOROPHYLLASE1 (CLH1) and CLH2 indicated that the known chlorophyllases are not responsible for the accumulation of chlorophyllide a in chlg-1. Taken together, our results show that chlorophyll synthase acts in a salvage pathway for chlorophyll biosynthesis by re-esterifying the chlorophyllide a produced during chlorophyll turnover.

Arabidopsis inositol pentakisphosphate 2-kinase, AtIPK1, is required for growth and modulates phosphate homeostasis at the transcriptional level.

Inositol hexakisphosphate (IP6 ) provides a phosphorous reservoir in plant seeds; in addition, along with its biosynthesis intermediates and derivatives, IP6 also plays important roles in diverse developmental and physiological processes. Disruption of the Arabidopsis inositol pentakisphosphate 2-kinase coding gene AtIPK1 was previously shown to reduce IP6 content in vegetative tissues and affect phosphate (Pi) sensing. Here we show that AtIPK1 is required for sustaining plant growth, as null mutants are non-viable. An incomplete loss-of-function mutant, atipk1-1, exhibited disturbed Pi homeostasis and overaccumulated Pi as a consequence of increased Pi uptake activity and root-to-shoot Pi translocation. The atipk1-1 mutants also showed a Pi deficiency-like root system architecture with reduced primary root and enhanced lateral root growth. Transcriptome analysis indicated that a subset of Pi starvation-responsive genes was transcriptionally perturbed in the atipk1-1 mutants and the expression of multiple genes involved in Pi uptake, allocation, and remobilization was increased. Genetic and transcriptional analyses suggest that disturbance of Pi homeostasis caused by atipk1 mutation involved components in addition to PHR1(-like) transcription factors. Notably, the transcriptional increase of a number of Pi starvation-responsive genes in the atipk1-1 mutants is correlated with the reduction of histone variant H2A.Z occupation in chromatin. The myo-inositol-1-phosphate synthase mutants, atmips1 and atmips2 with comparable reduction in vegetative IP6 to that in the atipk1-1 mutants did not overaccumulate Pi, suggesting that Pi homeostasis modulated by AtIPK1 is not solely attributable to IP6 level. This study reveals that AtIPK1 has important roles in growth and Pi homeostasis.

A positive feedback loop between HEAT SHOCK PROTEIN101 and HEAT STRESS-ASSOCIATED 32-KD PROTEIN modulates long-term acquired thermotolerance illustrating diverse heat stress responses in rice varieties.

Heat stress is an important factor that has a negative impact on rice (Oryza sativa) production. To alleviate this problem, it is necessary to extensively understand the genetic basis of heat tolerance and adaptability to heat stress in rice. Here, we report the molecular mechanism underlying heat acclimation memory that confers long-term acquired thermotolerance (LAT) in this monocot plant. Our results showed that a positive feedback loop formed by two heat-inducible genes, HEAT SHOCK PROTEIN101 (HSP101) and HEAT STRESS-ASSOCIATED 32-KD PROTEIN (HSA32), at the posttranscriptional level prolongs the effect of heat acclimation in rice seedlings. The interplay between HSP101 and HSA32 also affects basal thermotolerance of rice seeds. These findings are similar to those reported for the dicot plant Arabidopsis (Arabidopsis thaliana), suggesting a conserved function in plant heat stress response. Comparison between two rice cultivars, japonica Nipponbare and indica N22 showed opposite performance in basal thermotolerance and LAT assays. 'N22' seedlings have a higher basal thermotolerance level than cv Nipponbare and vice versa at the LAT level, indicating that these two types of thermotolerance can be decoupled. The HSP101 and HSA32 protein levels were substantially higher in cv Nipponbare than in cv N22 after a long recovery following heat acclimation treatment, at least partly explaining the difference in the LAT phenotype. Our results point out the complexity of thermotolerance diversity in rice cultivars, which may need to be taken into consideration when breeding for heat tolerance for different climate scenarios.

Common and distinct functions of Arabidopsis class A1 and A2 heat shock factors in diverse abiotic stress responses and development.

There are 21 heat shock factor (HSF) homologs in Arabidopsis (Arabidopsis thaliana), of which members of class A1 (HSFA1a/HSFA1b/HSFA1d/HSFA1e) play the major role in activating the transcription of heat-induced genes, including HSFA2. Once induced, HSFA2 becomes the dominant HSF and is able to form heterooligomeric complexes with HSFA1. However, whether HSFA2 could function independently as a transcription regulator in the absence of the HSFA1s was undetermined. To address this question, we introduced a Cauliflower mosaic virus 35S promoter:HSFA2 construct into hsfa1a/hsfa1b/hsfa1d/hsfa1e quadruple knockout (QK) and wild-type (Wt) backgrounds to yield transgenic lines A2QK and A2Wt, respectively. Constitutive expression of HSFA2 rescued the developmental defects of the QK mutant and promoted callus formation in A2QK, but not in A2Wt, after heat treatment. Transcriptome analysis showed that heat stress response genes are differentially regulated by the HSFA1s and HSFA2; the genes involved in metabolism and redox homeostasis are preferentially regulated by HSFA2, while HSFA1-preferring genes are enriched in transcription function. Ectopic expression of HSFA2 complemented the defects of QK in tolerance to different heat stress regimes, and to hydrogen peroxide, but not to salt and osmotic stresses. Furthermore, we showed that HSFA1a/HSFA1b/HSFA1d are involved in thermotolerance to mild heat stress at temperatures as low as 27°C. We also noticed subfunctionalization of the four Arabidopsis A1-type HSFs in diverse abiotic stress responses. Overall, this study reveals the overlapping and distinct functions of class A1 and A2 HSFs and may enable more precise use of HSFs in engineering stress tolerance in the future.

Interplay between heat shock proteins HSP101 and HSA32 prolongs heat acclimation memory posttranscriptionally in Arabidopsis.

Heat acclimation improves the tolerance of organisms to severe heat stress. Our previous work showed that in Arabidopsis (Arabidopsis thaliana), the "memory" of heat acclimation treatment decayed faster in the absence of the heat-stress-associated 32-kD protein HSA32, a heat-induced protein predominantly found in plants. The HSA32 null mutant attains normal short-term acquired thermotolerance but is defective in long-term acquired thermotolerance. To further explore this phenomenon, we isolated Arabidopsis defective in long-term acquired thermotolerance (dlt) mutants using a forward genetic screen. Two recessive missense alleles, dlt1-1 and dlt1-2, encode the molecular chaperone heat shock protein101 (HSP101). Results of immunoblot analyses suggest that HSP101 enhances the translation of HSA32 during recovery after heat treatment, and in turn, HSA32 retards the decay of HSP101. The dlt1-1 mutation has little effect on HSP101 chaperone activity and thermotolerance function but compromises the regulation of HSA32. In contrast, dlt1-2 impairs the chaperone activity and thermotolerance function of HSP101 but not the regulation of HSA32. These results suggest that HSP101 has a dual function, which could be decoupled by the mutations. Pulse-chase analysis showed that HSP101 degraded faster in the absence of HSA32. The autophagic proteolysis inhibitor E-64d, but not the proteasome inhibitor MG132, inhibited the degradation of HSP101. Ectopic expression of HSA32 confirmed its effect on the decay of HSP101 at the posttranscriptional level and showed that HSA32 was not sufficient to confer long-term acquired thermotolerance when the HSP101 level was low. Taken together, we propose that a positive feedback loop between HSP101 and HSA32 at the protein level is a novel mechanism for prolonging the memory of heat acclimation.

Recent gene duplication and subfunctionalization produced a mitochondrial GrpE, the nucleotide exchange factor of the Hsp70 complex, specialized in thermotolerance to chronic heat stress in Arabidopsis.

The duplication and divergence of heat stress (HS) response genes might help plants adapt to varied HS conditions, but little is known on the topic. Here, we examined the evolution and function of Arabidopsis (Arabidopsis thaliana) mitochondrial GrpE (Mge) proteins. GrpE acts as a nucleotide-exchange factor in the Hsp70/DnaK chaperone machinery. Genomic data show that AtMge1 and AtMge2 arose from a recent whole-genome duplication event. Phylogenetic analysis indicated that duplication and preservation of Mges occurred independently in many plant species, which suggests a common tendency in the evolution of the genes. Intron retention contributed to the divergence of the protein structure of Mge paralogs in higher plants. In both Arabidopsis and tomato (Solanum lycopersicum), Mge1 is induced by ultraviolet B light and Mge2 is induced by heat, which suggests regulatory divergence of the genes. Consistently, AtMge2 but not AtMge1 is under the control of HsfA1, the master regulator of the HS response. Heterologous expression of AtMge2 but not AtMge1 in the temperature-sensitive Escherichia coli grpE mutant restored its growth at 43°C. Arabidopsis T-DNA knockout lines under different HS regimes revealed that Mge2 is specifically required for tolerating prolonged exposure to moderately high temperature, as compared with the need of the heat shock protein 101 and the HS-associated 32-kD protein for short-term extreme heat. Therefore, with duplication and subfunctionalization, one copy of the Arabidopsis Mge genes became specialized in a distinct type of HS. We provide direct evidence supporting the connection between gene duplication and adaptation to environmental stress.

The role of class A1 heat shock factors (HSFA1s) in response to heat and other stresses in Arabidopsis.

In Arabidopsis, there are four homologs of class A1 heat shock factor (HSFA1) genes, which likely encode the master regulators of heat shock response (HSR). However, previous studies with double knockout (KO) mutants were unable to confirm this point probably due to functional redundancy. Here, we generated a quadruple KO (QK) and four triple KO mutants to dissect their functions. Our data show that members of the HSFA1 group not only play a pivotal role in HSR but also are involved in growth and development. Alterations in morphology and retardation in growth were observed in the quadruple but not in triple KO mutants. The basal and acquired thermotolerance capacity was dramatically decreased in the QK mutant but varied in triple KO mutants at different developmental stages. The transcriptomics profiles suggested that more than 65% of the heat stress (HS)-up-regulated genes were HSFA1 dependent. HSFA1s were also involved in the expression of several HS genes induced by H(2) O(2) , salt and mannitol, which is consistent with the increased sensitive phenotype of the QK mutant to the stress factors. In conclusion, the Arabidopsis HSFA1s function as the master regulators of HSR and participate as important components in other abiotic stress responses as well.

Chlorophyll dephytylation in chlorophyll metabolism: a simple reaction catalyzed by various enzymes.

Chlorophyll (Chl) is composed of a tetrapyrrole ring and a phytol tail, which facilitate light energy absorbance and assembly with photosynthetic protein complexes, respectively. Chl dephytylation, the hydrolytic removal of the phytol tail, is considered a pivotal step in diverse physiological processes, such as Chl salvage during repair of the photosystem, the Chl cycle in the adjustment of antenna size, and Chl breakdown in leaf senescence and fruit maturation. Moreover, phytol is a component of the tocopherols, a major form of vitamin E that is essential in the human diet. This phytol mostly comes from Chl hydrolysis. However, the authentic enzyme responsible for Chl dephytylation has proved elusive. CHLOROPHYLLASE (CLH) which was discovered over a century ago, was the first enzyme found to have dephytylation activity in vitro, but its role in Chl metabolism has been questioned and remains under debate. Recently, novel dephytylases, i.e., PHEOPHYTINASE (PPH) and CHLOROPHYLL DEPHYTYLASE1 (CLD1) have emerged from genetic studies, indicating that dephytylation in Chl catabolism involves different players and is more complicated than previously thought. Based on sequence homology, substrate specificity, and subcellular localization, CLH, PPH, and CLD1 belong to different types of dephytylase, which prompted us to re-examine the dilemmas and missing links that still exist in Chl metabolism. This review thus focuses on the hitherto unanswered questions involving the Chl dephytylation reaction by highlighting relevant literature, updating recent progress, and synthesizing ideas.

Temperature-induced lipocalin is required for basal and acquired thermotolerance in Arabidopsis.

Plant temperature-induced lipocalins (TILs) have been shown to be responsive to heat stress (HS), but the nature of this response was unknown. In this study, a reverse genetic approach was taken to elucidate the role of Arabidopsis TIL1 (At5g58070) in thermotolerance. A T-DNA knock-out line of TIL1 (til1-1) showed severe defects in basal (BT) and acquired thermotolerance (AT), which could be complemented by introducing the wild-type gene. However, over-expression of TIL1 did not significantly enhance thermotolerance in transgenic plants. TIL1 is peripherally associated with plasma membrane. Transcriptomic analysis showed that the heat shock response in til1-1 seedlings was about the same as in the wild-type plants except the expression of TIL1. The level of TIL1 did not affect the temperature threshold for heat shock protein induction. Ion leakage analysis revealed no significant difference in membrane stability between the wild-type and til1-1 seedlings. These results suggest that TIL1 is not involved in regulating membrane fluidity or stability. Nevertheless, the mutant plants were also more sensitive than the wild type to tert-butyl hydroperoxide, a reagent that induces lipid peroxidation. Taken together, these data indicate that TIL1 is an essential component for thermotolerance and probably functions by acting against lipid peroxidation induced by severe HS.

miR824/AGAMOUS-LIKE16 Module Integrates Recurring Environmental Heat Stress Changes to Fine-Tune Poststress Development.

Plant development is continually fine-tuned based on environmental factors. How environmental perturbations are integrated into the developmental programs and how poststress adaptation is regulated remains an important topic to dissect. Vegetative to reproductive phase change is a very important developmental transition that is complexly regulated based on endogenous and exogenous cues. Proper timing of flowering is vital for reproductive success. It has been shown previously that AGAMOUS LIKE 16 (AGL16), a MADS-box transcription factor negatively regulates flowering time transition through FLOWERING LOCUS T (FT), a central downstream floral integrator. AGL16 itself is negatively regulated by the microRNA miR824. Here we present a comprehensive molecular analysis of miR824/AGL16 module changes in response to mild and recurring heat stress. We show that miR824 accumulates gradually in response to heat due to the combination of transient transcriptional induction and posttranscriptional stability. miR824 induction requires heat shock cis-elements and activity of the HSFA1 family and HSFA2 transcription factors. Parallel to miR824 induction, its target AGL16 is decreased, implying direct causality. AGL16 posttranscriptional repression during heat stress, however, is more complex, comprising of a miRNA-independent, and a miR824-dependent pathway. We also show that AGL16 expression is leaf vein-specific and overlaps with miR824 (and FT) expression. AGL16 downregulation in response to heat leads to a mild derepression of FT. Finally, we present evidence showing that heat stress regulation of miR824/AGL16 is conserved within Brassicaceae. In conclusion, due to the enhanced post-transcriptional stability of miR824, stable repression of AGL16 is achieved following heat stress. This may serve to fine-tune FT levels and alter flowering time transition. Stress-induced miR824, therefore, can act as a "posttranscriptional memory factor" to extend the acute impact of environmental fluctuations in the poststress period.

Supraoptimal activity of CHLOROPHYLL DEPHYTYLASE1 results in an increase in tocopherol level in mature arabidopsis seeds.

Tocopherols are synthesized in photosynthetic organisms, playing a role in plant stress tolerance. Recent studies showed that the phytol moiety of tocopherols comes from the salvaged phytol chain during chlorophyll degradation. However, the enzyme(s) responsible for chlorophyll dephytylation remains unclear. Recently, we reported the identification and characterization of CHLOROPHYLL DEPHYTYLASE1 (CLD1) of Arabidopsis, suggesting its role in chlorophyll turnover at steady state. In this addendum to the report, we presented and discussed the results related to the function of CLD1 in tocopherol biosynthesis. The tocopherol levels in the mature seeds were not altered in the transgenic lines with reduced CLD1 expression but were moderately increased in the plants with supraoptimal CLD1 activity compared to wild type. These results suggest that manipulating CLD1 activity could affect tocopherol biosynthesis to a certain extent and that other dephytylating enzymes are sharing redundant function in contributing the phytol pool in plant cells.

The Cys-Arg/N-End Rule Pathway Is a General Sensor of Abiotic Stress in Flowering Plants.

Abiotic stresses impact negatively on plant growth, profoundly affecting yield and quality of crops. Although much is known about plant responses, very little is understood at the molecular level about the initial sensing of environmental stress. In plants, hypoxia (low oxygen, which occurs during flooding) is directly sensed by the Cys-Arg/N-end rule pathway of ubiquitin-mediated proteolysis, through oxygen-dependent degradation of group VII Ethylene Response Factor transcription factors (ERFVIIs) via amino-terminal (Nt-) cysteine [1, 2]. Using Arabidopsis (Arabidopsis thaliana) and barley (Hordeum vulgare), we show that the pathway regulates plant responses to multiple abiotic stresses. In Arabidopsis, genetic analyses revealed that response to these stresses is controlled by N-end rule regulation of ERFVII function. Oxygen sensing via the Cys-Arg/N-end rule in higher eukaryotes is linked through a single mechanism to nitric oxide (NO) sensing [3, 4]. In plants, the major mechanism of NO synthesis is via NITRATE REDUCTASE (NR), an enzyme of nitrogen assimilation [5]. Here, we identify a negative relationship between NR activity and NO levels and stabilization of an artificial Nt-Cys substrate and ERFVII function in response to environmental changes. Furthermore, we show that ERFVIIs enhance abiotic stress responses via physical and genetic interactions with the chromatin-remodeling ATPase BRAHMA. We propose that plants sense multiple abiotic stresses through the Cys-Arg/N-end rule pathway either directly (via oxygen sensing) or indirectly (via NO sensing downstream of NR activity). This single mechanism can therefore integrate environment and response to enhance plant survival.