CRISPR-Cas technology in corn: a new key to unlock genetic knowledge and create novel products.

Since its inception in 2012, CRISPR-Cas technologies have taken the life science community by storm. Maize genetics research is no exception. Investigators around the world have adapted CRISPR tools to advance maize genetics research in many ways. The principle application has been targeted mutagenesis to confirm candidate genes identified using map-based methods. Researchers are also developing tools to more effectively apply CRISPR-Cas technologies to maize because successful application of CRISPR-Cas relies on target gene identification, guide RNA development, vector design and construction, CRISPR-Cas reagent delivery to maize tissues, and plant characterization, each contributing unique challenges to CRISPR-Cas efficacy. Recent advances continue to chip away at major barriers that prevent more widespread use of CRISPR-Cas technologies in maize, including germplasm-independent delivery of CRISPR-Cas reagents and production of high-resolution genomic data in relevant germplasm to facilitate CRISPR-Cas experimental design. This has led to the development of novel breeding tools to advance maize genetics and demonstrations of how CRISPR-Cas technologies might be used to enhance maize germplasm. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01200-9.

Multiple insertions of COIN, a novel maize Foldback transposable element, in the Conring gene cause a spontaneous progressive cell death phenotype.

Similar progressive leaf lesion phenotypes, named conring for "concentric ring," were identified in 10 independently derived maize lines. Complementation and mapping experiments indicated that the phenotype had the same genetic basis in each line - a single recessive gene located in a 1.1-Mb region on chromosome 2. Among the 15 predicted genes in this interval, Zm00001d003866 (subsequently renamed Conring or Cnr) had insertions of four related 138 bp transposable element (TE) sequences at precisely the same site in exon 4 in nine of the 10 cnr alleles. The 10th cnr allele had a distinct insertion of 226 bp of in exon 3. Genetic evidence suggested that the 10 cnr alleles were independently derived, and arose during the derivation of each line. The four TEs, named COINa (for COnring INsertion) through COINd, have not been previously characterized and consist entirely of imperfect 69-bp terminal inverted repeats characteristic of the Foldback class of TEs. They belong to three clades of a family of maize TEs comprising hundreds of sequences in the genome of the B73 maize line. COIN elements preferentially insert at TNA sequences with a preference for C and G nucleotides in the immediately flanking 5' and 3' regions, respectively. They produce a three-base target site duplication and do not have homology to other characterized TEs. We propose that Cnr is an unstable gene that is mutated insertionally at high frequency, most commonly due to COIN element insertions at a specific site in the gene.

Control of meristem determinacy by trehalose 6-phosphate phosphatases is uncoupled from enzymatic activity.

Meristem fate is regulated by trehalose 6-phosphate phosphatases (TPPs), but their mechanism of action remains mysterious. Loss of the maize TPPs RAMOSA3 and TPP4 leads to reduced meristem determinacy and more inflorescence branching. However, analysis of an allelic series revealed no correlation between enzymatic activity and branching, and a catalytically inactive version of RA3 complements the ra3 mutant. Together with their nuclear localization, these findings suggest a moonlighting function for TPPs.

Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis.

The phytohormone cytokinin has been shown to affect many aspects of plant development ranging from the regulation of the shoot apical meristem to leaf senescence. However, some studies have reported contradictory effects of cytokinin on leaf physiology. Therefore cytokinin treatments cause both chlorosis and increased greening and both lead to decrease or increase in cell size. To elucidate this multifaceted role of cytokinin in leaf development, we have employed a system of temporal controls over the cytokinin pool and investigated the consequences of modulated cytokinin levels in the third leaf of Arabidopsis. We show that, at the cell proliferation phase, cytokinin is needed to maintain cell proliferation by blocking the transition to cell expansion and the onset of photosynthesis. Transcriptome profiling revealed regulation by cytokinin of a gene suite previously shown to affect cell proliferation and expansion and thereby a molecular mechanism by which cytokinin modulates a molecular network underlying the cellular responses. During the cell expansion phase, cytokinin stimulates cell expansion and differentiation. Consequently, a cytokinin excess at the cell expansion phase results in an increased leaf and rosette size fueled by higher cell expansion rate, yielding higher shoot biomass. Proteome profiling revealed the stimulation of primary metabolism by cytokinin, in line with an increased sugar content that is expected to increase turgor pressure, representing the driving force of cell expansion. Therefore, the developmental timing of cytokinin content fluctuations, together with a tight control of primary metabolism, is a key factor mediating transitions from cell proliferation to cell expansion in leaves.

Altered expression of maize PLASTOCHRON1 enhances biomass and seed yield by extending cell division duration.

Maize is the highest yielding cereal crop grown worldwide for grain or silage. Here, we show that modulating the expression of the maize PLASTOCHRON1 (ZmPLA1) gene, encoding a cytochrome P450 (CYP78A1), results in increased organ growth, seedling vigour, stover biomass and seed yield. The engineered trait is robust as it improves yield in an inbred as well as in a panel of hybrids, at several locations and over multiple seasons in the field. Transcriptome studies, hormone measurements and the expression of the auxin responsive DR5rev:mRFPer marker suggest that PLA1 may function through an increase in auxin. Detailed analysis of growth over time demonstrates that PLA1 stimulates the duration of leaf elongation by maintaining dividing cells in a proliferative, undifferentiated state for a longer period of time. The prolonged duration of growth also compensates for growth rate reduction caused by abiotic stresses.

Time of day determines Arabidopsis transcriptome and growth dynamics under mild drought.

Drought stress is a major problem for agriculture worldwide, causing significant yield losses. Plants have developed highly flexible mechanisms to deal with drought, including organ- and developmental stage-specific responses. In young leaves, growth is repressed as an active mechanism to save water and energy, increasing the chances of survival but decreasing yield. Despite its importance, the molecular basis for this growth inhibition is largely unknown. Here, we present a novel approach to explore early molecular mechanisms controlling Arabidopsis leaf growth inhibition following mild drought. We found that growth and transcriptome responses to drought are highly dynamic. Growth was only repressed by drought during the day, and our evidence suggests that this may be due to gating by the circadian clock. Similarly, time of day strongly affected the extent, specificity, and in certain cases even direction of drought-induced changes in gene expression. These findings underscore the importance of taking into account diurnal patterns to understand stress responses, as only a small core of drought-responsive genes are affected by drought at all times of the day. Finally, we leveraged our high-resolution data to demonstrate that phenotypic and transcriptome responses can be matched to identify putative novel regulators of growth under mild drought.

The ETHYLENE RESPONSE FACTORs ERF6 and ERF11 Antagonistically Regulate Mannitol-Induced Growth Inhibition in Arabidopsis.

Leaf growth is a tightly regulated and complex process, which responds in a dynamic manner to changing environmental conditions, but the mechanisms that reduce growth under adverse conditions are rather poorly understood. We previously identified a growth inhibitory pathway regulating leaf growth upon exposure to a low concentration of mannitol and characterized the ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 transcription factor ERF6 as a central activator of both leaf growth inhibition and induction of stress tolerance genes. Here, we describe the role of the transcriptional repressor ERF11 in relation to the ERF6-mediated stress response in Arabidopsis (Arabidopsis thaliana). Using inducible overexpression lines, we show that ERF6 induces the expression of ERF11. ERF11 in turn molecularly counteracts the action of ERF6 and represses at least some of the ERF6-induced genes by directly competing for the target gene promoters. As a phenotypical consequence of the ERF6-ERF11 antagonism, the extreme dwarfism caused by ERF6 overexpression is suppressed by overexpression of ERF11. Together, our data demonstrate that dynamic mechanisms exist to fine-tune the stress response and that ERF11 counteracts ERF6 to maintain a balance between plant growth and stress defense.

What Is Stress? Dose-Response Effects in Commonly Used in Vitro Stress Assays.

In vitro stress assays are commonly used to study the responses of plants to abiotic stress and to assess stress tolerance. A literature review reveals that most studies use very high stress levels and measure criteria such as germination, plant survival, or the development of visual symptoms such as bleaching. However, we show that these parameters are indicators of very severe stress, and such studies thus only provide incomplete information about stress sensitivity in Arabidopsis (Arabidopsis thaliana). Similarly, transcript analysis revealed that typical stress markers are only induced at high stress levels in young seedlings. Therefore, tools are needed to study the effects of mild stress. We found that the commonly used stress-inducing agents mannitol, sorbitol, NaCl, and hydrogen peroxide impact shoot growth in a highly specific and dose-dependent way. Therefore, shoot growth is a sensitive, relevant, and easily measured phenotype to assess stress tolerance over a wide range of stress levels. Finally, our data suggest that care should be taken when using mannitol as an osmoticum.

Poly(ADP-ribose)polymerase activity controls plant growth by promoting leaf cell number.

A changing global environment, rising population and increasing demand for biofuels are challenging agriculture and creating a need for technologies to increase biomass production. Here we demonstrate that the inhibition of poly (ADP-ribose) polymerase activity is a promising technology to achieve this under non-stress conditions. Furthermore, we investigate the basis of this growth enhancement via leaf series and kinematic cell analysis as well as single leaf transcriptomics and plant metabolomics under non-stress conditions. These data indicate a regulatory function of PARP within cell growth and potentially development. PARP inhibition enhances growth of Arabidopsis thaliana by enhancing the cell number. Time course single leaf transcriptomics shows that PARP inhibition regulates a small subset of genes which are related to growth promotion, cell cycle and the control of metabolism. This is supported by metabolite analysis showing overall changes in primary and particularly secondary metabolism. Taken together the results indicate a versatile function of PARP beyond its previously reported roles in controlling plant stress tolerance and thus can be a useful target for enhancing biomass production.

Transcriptional coordination between leaf cell differentiation and chloroplast development established by TCP20 and the subgroup Ib bHLH transcription factors.

The establishment of the photosynthetic apparatus during chloroplast development creates a high demand for iron as a redox metal. However, iron in too high quantities becomes toxic to the plant, thus plants have evolved a complex network of iron uptake and regulation mechanisms. Here, we examined whether four of the subgroup Ib basic helix-loop-helix transcription factors (bHLH38, bHLH39, bHLH100, bHLH101), previously implicated in iron homeostasis in roots, also play a role in regulating iron metabolism in developing leaves. These transcription factor genes were strongly up-regulated during the transition from cell proliferation to expansion, and thus sink-source transition, in young developing leaves of Arabidopsis thaliana. The four subgroup Ib bHLH genes also showed reduced expression levels in developing leaves of plants treated with norflurazon, indicating their expression was tightly linked to the onset of photosynthetic activity in young leaves. In addition, we provide evidence for a mechanism whereby the transcriptional regulators SAC51 and TCP20 antagonistically regulate the expression of these four subgroup Ib bHLH genes. A loss-of-function mutant analysis also revealed that single mutants of bHLH38, bHLH39, bHLH100, and bHLH101 developed smaller rosettes than wild-type plants in soil. When grown in agar plates with reduced iron concentration, triple bhlh39 bhlh100 bhlh101 mutant plants were smaller than wild-type plants. However, measurements of the iron content in single and multiple subgroup Ib bHLH genes, as well as transcript profiling of iron response genes during early leaf development, do not support a role for bHLH38, bHLH39, bHLH100, and bHLH101 in iron homeostasis during early leaf development.

Gibberellins and DELLAs: central nodes in growth regulatory networks.

Gibberellins (GAs) are growth-promoting phytohormones that were crucial in breeding improved semi-dwarf varieties during the green revolution. However, the molecular basis for GA-induced growth stimulation is poorly understood. In this review, we use light-regulated hypocotyl elongation as a case study, combined with a meta-analysis of available transcriptome data, to discuss the role of GAs as central nodes in networks connecting environmental inputs to growth. These networks are highly tissue-specific, with dynamic and rapid regulation that mostly occurs at the protein level, directly affecting the activity and transcription of effectors. New systems biology approaches addressing the role of GAs in growth should take these properties into account, combining tissue-specific interactomics, transcriptomics and modeling, to provide essential knowledge to fuel a second green revolution.

The agony of choice: how plants balance growth and survival under water-limiting conditions.

When confronted with water limitation, plants actively reprogram their metabolism and growth. Recently, it has become clear that growing tissues show specific and highly dynamic responses to drought, which differ from the well-studied responses in mature tissues. Here, we provide an overview of recent advances in understanding shoot growth regulation in water-limiting conditions. Of special interest is the balance between maintained growth and competitiveness on the one hand and ensured survival on the other hand. A number of master regulators controlling this balance have been identified, such as DELLAs and APETALA2/ETHYLENE RESPONSE FACTOR-type transcription factors. The possibilities of engineering or breeding crops that maintain growth in periods of mild drought, while still being able to activate protective tolerance mechanisms, are discussed.

Ethylene Response Factor6 acts as a central regulator of leaf growth under water-limiting conditions in Arabidopsis.

Leaf growth is a complex developmental process that is continuously fine-tuned by the environment. Various abiotic stresses, including mild drought stress, have been shown to inhibit leaf growth in Arabidopsis (Arabidopsis thaliana), but the underlying mechanisms remain largely unknown. Here, we identify the redundant Arabidopsis transcription factors ETHYLENE RESPONSE FACTOR5 (ERF5) and ERF6 as master regulators that adapt leaf growth to environmental changes. ERF5 and ERF6 gene expression is induced very rapidly and specifically in actively growing leaves after sudden exposure to osmotic stress that mimics mild drought. Subsequently, enhanced ERF6 expression inhibits cell proliferation and leaf growth by a process involving gibberellin and DELLA signaling. Using an ERF6-inducible overexpression line, we demonstrate that the gibberellin-degrading enzyme GIBBERELLIN 2-OXIDASE6 is transcriptionally induced by ERF6 and that, consequently, DELLA proteins are stabilized. As a result, ERF6 gain-of-function lines are dwarfed and hypersensitive to osmotic stress, while the growth of erf5erf6 loss-of-function mutants is less affected by stress. Besides its role in plant growth under stress, ERF6 also activates the expression of a plethora of osmotic stress-responsive genes, including the well-known stress tolerance genes STZ, MYB51, and WRKY33. Interestingly, activation of the stress tolerance genes by ERF6 occurs independently from the ERF6-mediated growth inhibition. Together, these data fit into a leaf growth regulatory model in which ERF5 and ERF6 form a missing link between the previously observed stress-induced 1-aminocyclopropane-1-carboxylic acid accumulation and DELLA-mediated cell cycle exit and execute a dual role by regulating both stress tolerance and growth inhibition.

DELLA signaling mediates stress-induced cell differentiation in Arabidopsis leaves through modulation of anaphase-promoting complex/cyclosome activity.

Drought is responsible for considerable yield losses in agriculture due to its detrimental effects on growth. Drought responses have been extensively studied, but mostly on the level of complete plants or mature tissues. However, stress responses were shown to be highly tissue and developmental stage specific, and dividing tissues have developed unique mechanisms to respond to stress. Previously, we studied the effects of osmotic stress on dividing leaf cells in Arabidopsis (Arabidopsis thaliana) and found that stress causes early mitotic exit, in which cells end their mitotic division and start endoreduplication earlier. In this study, we analyzed this phenomenon in more detail. Osmotic stress induces changes in gibberellin metabolism, resulting in the stabilization of DELLAs, which are responsible for mitotic exit and earlier onset of endoreduplication. Consequently, this response is absent in mutants with altered gibberellin levels or DELLA activity. Mitotic exit and onset of endoreduplication do not correlate with an up-regulation of known cell cycle inhibitors but are the result of reduced levels of DP-E2F-LIKE1/E2Fe and UV-B-INSENSITIVE4, both inhibitors of the developmental transition from mitosis to endoreduplication by modulating anaphase-promoting complex/cyclosome activity, which are down-regulated rapidly after DELLA stabilization. This work fits into an emerging view of DELLAs as regulators of cell division by regulating the transition to endoreduplication and differentiation.

Pause-and-stop: the effects of osmotic stress on cell proliferation during early leaf development in Arabidopsis and a role for ethylene signaling in cell cycle arrest.

Despite its relevance for agricultural production, environmental stress-induced growth inhibition, which is responsible for significant yield reductions, is only poorly understood. Here, we investigated the molecular mechanisms underlying cell cycle inhibition in young proliferating leaves of the model plant Arabidopsis thaliana when subjected to mild osmotic stress. A detailed cellular analysis demonstrated that as soon as osmotic stress is sensed, cell cycle progression rapidly arrests, but cells are kept in a latent ambivalent state allowing a quick recovery (pause). Remarkably, cell cycle arrest coincides with an increase in 1-aminocyclopropane-1-carboxylate levels and the activation of ethylene signaling. Our work showed that ethylene acts on cell cycle progression via inhibition of cyclin-dependent kinase A activity independently of EIN3 transcriptional control. When the stress persists, cells exit the mitotic cell cycle and initiate the differentiation process (stop). This stop is reflected by early endoreduplication onset, in a process independent of ethylene. Nonetheless, the potential to partially recover the decreased cell numbers remains due to the activity of meristemoids. Together, these data present a conceptual framework to understand how environmental stress reduces plant growth.

Developmental stage specificity and the role of mitochondrial metabolism in the response of Arabidopsis leaves to prolonged mild osmotic stress.

When subjected to stress, plants reprogram their growth by largely unknown mechanisms. To provide insights into this process, the growth of Arabidopsis (Arabidopsis thaliana) leaves that develop under mild osmotic stress was studied. Early during leaf development, cell number and size were reduced by stress, but growth was remarkably adaptable, as division and expansion rates were identical to controls within a few days of leaf initiation. To investigate the molecular basis of the observed adaptability, leaves with only proliferating, exclusively expanding, and mature cells were analyzed by transcriptomics and targeted metabolomics. The stress response measured in growing and mature leaves was largely distinct; several hundred transcripts and multiple metabolites responded exclusively in the proliferating and/or expanding leaves. Only a few genes were differentially expressed across the three stages. Data analysis showed that proliferation and expansion were regulated by common regulatory circuits, involving ethylene and gibberellins but not abscisic acid. The role of ethylene was supported by the analysis of ethylene-insensitive mutants. Exclusively in proliferating cells, stress induced genes of the so-called "mitochondrial dysfunction regulon," comprising alternative oxidase. Up-regulation for eight of these genes was confirmed with promoter:beta-glucuronidase reporter lines. Furthermore, mitochondria of stress-treated dividing cells were morphologically distinct from control ones, and growth of plants overexpressing the alternative oxidase gene was more tolerant to osmotic and drought stresses. Taken together, our data underline the value of analyzing stress responses in development and demonstrate the importance of mitochondrial respiration for sustaining cell proliferation under osmotic stress conditions.