Primary carbohydrate metabolism genes participate in heat-stress memory at the shoot apical meristem of Arabidopsis thaliana

(Molecular Plant 14,1508-1524,September 62021)After the original publication of the above article,attentive readers identified errors in Figure 4H that needed correction as follows.Figure 4H contained the erroneous labels“10 DAP(7 DAT),”which should have each read,“7 DAP(4 DAT).”To avoid any confusion and ensure consistency across our figures,we modified some of the Col-0 images in a corrected version of Figure 4H below to resemble the exact same Col-0 images as in Figure 1C.The details of the changes are as follows:rotation(Col-0 Primed,all timepoints;Col-0 Triggered,3 DAP and 5 DAP;Col-0 Primed/triggered,3 DAP and 5 DAP),replacement(Col-0 Triggered,7 DAP),and different cropping(Col-0 Primed/triggered,7 DAP).

The central role of stem cells in determining plant longevity variation

Vascular plants display a huge variety of longevity patterns,from a few weeks for several annual species up to thousands of years for some perennial species.Understanding how longevity variation is structured has long been considered a fundamental aspect of the life sciences in view of evolution,species distribution,and adaptation to diverse environments.Unlike animals,whose organs are typically formed during embryogenesis,vascular plants manage to extend their life by continuously producing new tissues and organs in apical and lateral directions via proliferation of stem cells located within specialized tissues called meristems.Stem cells are the main source of plant longevity.Variation in plant longevity is highly dependent on the activity and fate identity of stem cells.Multiple developmental factors determine how stem cells contribute to variation in plant longevity.In this review,we provide an overview of the genetic mechanisms,hormonal signaling,and environmental factors involved in controlling plant longevity through long-term maintenance of stem cell fate identity.

Growth-Regulating Factors （GRFs）： A Small Transcription Factor Family with Important Functions in Plant Biology

Growth-regulating factors （GRFs） are plant-specific transcription factors that were originally identified for their roles in stem and leaf development, but recent studies highlight them to be similarly important for other central developmental processes including flower and seed formation, root development, and the coordination of growth processes under adverse environmental conditions. The expression of several GRFs is controlled by microRNA miR396, and the GRF-miRNA396 regulatory module appears to be central to several of these processes. In addition, transcription factors upstream of GRFs and miR396 have been discovered, and gradually downstream target genes of GRFs are being unraveled. Here, we review the current knowledge of the biological functions performed by GRFs and survey available molecular data to illustrate how they exert their roles at the cellular level.

ORS1, an H2O2-Responsive NAC Transcription Factor, Controls Senescence in Arabidopsis thaliana

We report here that ORS1, a previously uncharacterized member of the NAC transcription factor family, controls leaf senescence in Arabidopsis thaliana. Overexpression of ORS1 accelerates senescence in transgenic plants, whereas its inhibition delays it. Genes acting downstream of ORS1 were identified by global expression analysis using transgenic plants producing dexamethasone-inducible ORSl-GR fusion protein. Of the 42 up-regulated genes, 30 （-70%） were pre- viously shown to be up-regulated during age-dependent senescence, We also observed that 32 （-76%） of the ORSl-dependent genes were induced by long-term （4 d）, but not short-term （6 h） salinity stress （150 mM NaCI）. Furthermore, expression of 16 and 24 genes, respectively, was induced after 1 and 5 h of treatment with hydrogen peroxide （H2O2）, a reactive oxygen species known to accumulate during salinity stress. ORS1 itself was found to be rapidly and strongly induced by H2O2 treatment in both leaves and roots. Using in vitro binding site selection, we determined the preferred binding motif of ORS1 and found it to be present in half of the ORSl-dependent genes. ORS1 is a paralog of ORE1/ ANACO92/AtNAC2, a previously reported regulator of leaf senescence. Phylogenetic footprinting revealed evolutionary conservation of the ORS1 and ORE1 promoter sequences in different Brassicaceae species, indicating strong positive selection acting on both genes. We conclude that ORS1, similarly to ORE1, triggers expression of senescence-associated genes through a regulatory network that may involve cross-talk with saltand H2O2-dependent signaling pathways.

NAC Transcription Factor ORE1 and Senescence- Induced BIFUNCTIONAL NUCLEASE1 （BFN1） Constitute a Regulatory Cascade in Arabidopsis

Senescence is a highly regulated process that involves the action of a large number of transcription factors. The NAC transcription factor ORE1 （ANAC092） has recently been shown to play a critical role in positively controlling senescence in Arabidopsis thaliana; however, no direct target gene through which it exerts its molecular function has been identified previously. Here, we report that BIFUNCTIONAL NUCLEASE1 （BFN1）, a well-known senescence-enhanced gene, is directly regulated by ORE1. We detected elevated expression of BFN1 already 2 h after induction of ORE1 in estradiol-inducible ORE1 overexpression lines and 6 h after transfection of Arabidopsis mesophyll cell protoplasts with a 35S：ORE1 construct, ORE1 and BFN1 expression patterns largely overlap, as shown by promoter-reporter gene （GUS） fusions, while BFN1 expression in senescent leaves and the abscission zones of maturing flower organs was virtually absent in ore1 mutant background. In vitro binding site assays revealed a bipartite ORE1 binding site, similar to that of ORS1, a paralog of ORE1. A bipartite ORE1 binding site was identified in the BFN1 promoter; mutating the cis-element within the context of the full-length BFN1 promoter drastically reduced OREl-mediated transactivation capacity in tran- siently transfected Arabidopsis mesophyll cell protoplasts. Furthermore, chromatin immunoprecipitation （CHIP） demon- strates in vivo binding of ORE1 to the BFN1 promoter. We also demonstrate binding of ORE1 in vivo to the promoters of two other senescence-associated genes, namely SAG29/SWEET15 and SINA1, supporting the central role of ORE1 during senescence.

The LysM Receptor-Like Kinase LysM RLK1 Is Required to Activate Defense and Abiotic-Stress Responses Induced by Overexpression of Fungal Chitinases in Arabidopsis Plants

Application of crab shell chitin or pentamer chitin oligosaccharide to Arabidopsis seedlings increased toler- ance to salinity in wild-type but not in knockout mutants of the LysM Receptor-Like Kinasel （CERK1/LysM RLK1） gene, known to play a critical role in signaling defense responses induced by exogenous chitin. Arabidopsis plants overexpress- ing the endochitinase chit36 and hexoaminidase excyl genes from the fungus Trichoderma asperelleoides T203 showed increased tolerance to salinity, heavy-metal stresses, and Botrytis cinerea infection. Resistant lines, overexpressing fungal chitinases at different levels, were outcrossed to lysm rlkl mutants. Independent homozygous hybrids lost resistance to biotic and abiotic stresses, despite enhanced chitinase activity. Expression analysis of 270 stress-related genes, including those induced by reactive oxygen species （ROS） and chitin, revealed constant up-regulation （at least twofold） of 10 genes in the chitinase-overexpressing line and an additional 76 salt-induced genes whose expression was not elevated in the lysm rlkl knockout mutant or the hybrids harboring the mutation. These findings elucidate that chitin-induced signaling mediated by LysM RLK1 receptor is not limited to biotic stress response but also encompasses abiotic-stress signaling and can be conveyed by ectopic expression of chitinases in plants.

Distributed Differences Structures Underlie Gating between the Kin Channel KAT1 and the Kout Channel SKOR

The family of voltage-gated （Shaker-like） potassium channels in plants includes both inward-rectifying （Kin） channels that allow plant cells to accumulate K＋ and outward-rectifying （Kout） channels that mediate K＋ efflux. Despite their dose structural similarities, Kin and Kout channels differ in their gating sensitivity towards voltage and the extracellular K＋ concentration. We have carried out a systematic program of domain swapping between the Kout channel SKOR and the Kin channel KAT1 to examine the impacts on gating of the pore regions, the S4, S5, and the S6 helices. We found that, in particular, the N-terminal part of the S5 played a critical role in KAT1 and SKOR gating. Our findings were supported by molecular dynamics of KAT1 and SKOR homology models. In silico analysis revealed that during channel opening and closing, displacement of certain residues, especially in the S5 and S6 segments, is more pronounced in KAT1 than in SKOR. From our analysis of the S4-S6 region, we conclude that gating （and K＋-sensing in SKOR） depend on a number of structural elements that are dispersed over this -145-residue sequence and that these place additional constraints on configurational rearrangement of the channels during gating.

Primary carbohydrate metabolism genes participate in heat-stress memory at the shoot apical meristem of Arabidopsis thaliana

In plants, the shoot apical meristem (SAM) is essential for the growth of aboveground organs. However, little is known about its molecular responses to abiotic stresses. Here, we show that the SAM of Arabidopsis thaliana displays an autonomous heat-stress (HS) memory of a previous non-lethal HS, allowing the SAM to regain growth after exposure to an otherwise lethal HS several days later. Using RNA sequencing, we identified genes participating in establishing the SAM's HS transcriptional memory, including the stem cell (SC) regulators CLAVATA1 (CLV1) and CLV3, HEAT SHOCK PROTEIN 17.6A (HSP17.6A), and the primary carbohydrate metabolism gene FRUCTOSE-BISPHOSPHATE ALDOLASE 6 (FBA6). We demonstrate that sugar availability is essential for survival of plants at high temperature. HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2A) directly regulates the expression of HSP17.6A and FBA6 by binding to the heat-shock elements in their promoters, indicating that HSFA2 is required for transcriptional activation of SAM memory genes. Collectively, these findings indicate that plants have evolved a sophisticated protection mechanism to maintain SCs and, hence, their capacity to re-initiate shoot growth after stress release.

A step-by-step protocol for formaldehyde-assisted isolation of regulatory elements from Arabidopsis thaliana

The control of gene expression by transcriptional regulators and other types of functional y relevant DNA transactions such as chromatin remodeling and replication underlie a vast spectrum of biological processes in al organisms. DNA transactions require the control ed interaction of proteins with DNA sequence motifs which are often located in nucleosome-depleted regions （NDRs） of the chromatin. Formaldehyde-assisted isolation of regulatory elements （FAIRE） has been established as an easy-to-implement method for the isolation of NDRs from a number of eukaryotic organisms, and it has been successful y employed for the discovery of new regulatory segments in genomic DNA from, for example, yeast, Drosophila, and humans. Until today, however, FAIRE has only rarely been employed in plant research and currently no detailed FAIRE protocol for plants has been published. Here, we provide a step-by-step FAIRE protocol for NDR discovery in Arabidopsis thaliana. We demonstrate that NDRs isolated from plant chromatin are readily amenable to quantitative polymerase chain reaction and next-generation sequencing. Only minor modification of the FAIRE protocol wil be needed to adapt it to other plants, thus facilitating the global inventory of regulatory regions across species.

The HB40-JUB1 transcriptional regulatory network controls gibberellin homeostasis in Arabidopsis

The gibberellins(GAs)are phytohormones that play fundamental roles in almost every aspect of plant growth and development.Although GA biosynthetic and signaling pathways are well understood,the mechanisms that control GA homeostasis remain largely unclear in plants.Here,we demonstrate that the homeobox transcription factor(TF)HB40 of the HD-Zip family regulates GA content at two additive con-trol levels in Arabidopsis thaliana.We show that HB40 expression is induced by GA and in turn reduces the levels of endogenous bioactive GAs by simultaneously reducing GA biosynthesis and increasing GA deac-tivation.Consistently,HB40 overexpression leads to typical GA-deficiency traits,such as small rosettes,reduced plant height,delayed flowering,and male sterility.By contrast,a loss-of-function hb40 mutation enhances GA-controlled growth.Genome-wide RNA sequencing combined with molecular-genetic ana-lyses revealed that HB40 directly activates the transcription of JUNGBRUNNEN1(JJUB1),a key TF that re-presses growth by suppressing GA biosynthesis and signaling.HB40 also activates genes encoding GA 2-oxidases(GA2oxs),which are major GA-catabolic enzymes.The effect of HB40 on plant growth is ultimately mediated through the induction of nuclear growth-repressing DELLA proteins.Collectively,our results reveal the important role of the HB40-JUB1 regulatory network in controlling GA homeostasis during plant growth.

The plant circadian clock regulates autophagy rhythm through transcription factor LUX ARRHYTHMO

Autophagy is an evolutionarily conserved degradation pathway in eukaryotes;it plays a critical role in nutritional stress tolerance.The circadian clock is an endogenous timekeeping system that generates biological rhythms to adapt to daily changes in the environment.Accumulating evidence indicates that the circadian clock and autophagy are intimately interwoven in animals.However,the role of the circadian clock in regulating autophagy has been poorly elucidated in plants.Here,we show that autophagy exhibits a robust circadian rhythm in both light/dark cycle(LD)and in constant light(LL)in Arabidopsis.However,autophagy rhythm showed a different pattem with a phase-advance shift and a lower amplitude in LL compared to LD.Moreover,mutation of the transcription factor LUX ARRHYTHMO(LUX)removed autophagy rhythm in LL and led to an enhanced amplitude in LD.LUX represses expression of the core autophagy genes ATG2,ATG8 a,and ATG11 by directly binding to their promoters.Phenotypic analysis revealed that LUX is responsible for improved resistance of plants to carbon starvation,which is dependent on moderate autophagy activity.Comprehensive transcriptomic analysis revealed that the autophagy rhythm is ubiquitous in plants.Taken together,our findings demonstrate that the LUXmediated circadian clock regulates plant autophagy rhythms.