Leaf growth in dicots and monocots : so different yet so alike

In plants, most organs grow post-embryonically through cell division and cell expansion. The coordination of these two growth processes is generally considered to be different between dicots and monocots. In dicot plants, such as the model plant Arabidopsis, leaf growth is most often described as being temporally regulated with cell division ceasing earlier at the tip and continuing longer at the base of the leaf. Conversely, in monocot leaves, the organization of the growth processes is rather viewed as spatially regulated with dividing cells at the base of the leaf, followed by expanding cells and finally mature cells at the tip. As our understanding of the leaf growth processes in the two major classes of flowering plants expands, it becomes increasingly clear that the regulation of the growth processes is to a great extent conserved between dicots and monocots. In this review, we highlight how the temporal and spatial organization of cell division and cell expansion takes place in both dicot and monocot leaves. We also show that there are similarities in the molecular wiring that coordinates these two processes during leaf development

Overexpression of GA20-OXIDASE1 impacts plant height, biomass allocation and saccharification efficiency in maize

Increased biomass yield and quality are of great importance for the improvement of feedstock for the biorefinery. For the production of bioethanol, both stem biomass yield and the conversion efficiency of the polysaccharides in the cell wall to fermentable sugars are of relevance. Increasing the endogenous levels of gibberellic acid (GA) by ectopic expression of GA20-OXIDASE1 (GA20-OX1), the rate-limiting step in GA biosynthesis, is known to affect cell division and cell expansion, resulting in larger plants and organs in several plant species. In this study, we examined biomass yield and quality traits of maize plants overexpressing GA20-OX1 (GA20-OX1). GA20-OX1 plants accumulated more vegetative biomass than control plants in greenhouse experiments, but not consistently over two years of field trials. The stems of these plants were longer but also more slender. Investigation of GA20-OX1 biomass quality using biochemical analyses showed the presence of more cellulose, lignin and cell wall residue. Cell wall analysis as well as expression analysis of lignin biosynthetic genes in developing stems revealed that cellulose and lignin were deposited earlier in development. Pretreatment of GA20-OX1 biomass with NaOH resulted in a higher saccharification efficiency per unit of dry weight, in agreement with the higher cellulose content. On the other hand, the cellulose-to-glucose conversion was slower upon HCl or hot-water pretreatment, presumably due to the higher lignin content. This study showed that biomass yield and quality traits can be interconnected, which is important for the development of future breeding strategies to improve lignocellulosic feedstock for bioethanol production

LEAF-E: a tool to analyze grass leaf growth using function fitting

In grasses, leaf growth is often monitored to gain insights in growth processes, biomass accumulation, regrowth after cutting, etc. To study the growth dynamics of the grass leaf, its length is measured at regular time intervals to derive the leaf elongation rate (LER) profile over time. From the LER profile, parameters such as maximal LER and leaf elongation duration (LED), which are essential for detecting inter-genotype growth differences and/or quantifying plant growth responses to changing environmental conditions, can be determined. As growth is influenced by the circadian clock and, especially in grasses, changes in environmental conditions such as temperature and evaporative demand, the LER profiles show considerable experimental variation and thus often do not follow a smooth curve. Hence it is difficult to quantify the duration and timing of growth. For these reasons, the measured data points should be fitted using a suitable mathematical function, such as the beta sigmoid function for leaf elongation. In the context of high-throughput phenotyping, we implemented the fitting of leaf growth measurements into a user-friendly Microsoft Excel-based macro, a tool called LEAF-E. LEAF-E allows to perform non-linear regression modeling of leaf length measurements suitable for robust and automated extraction of leaf growth parameters such as LER and LED from large datasets. LEAF-E is particularly useful to quantify the timing of leaf growth, which forms an important added value for detecting differences in leaf growth development. We illustrate the broad application range of LEAF-E using published and unpublished data sets of maize, Miscanthus spp. and Brachypodium distachyon, generated in independent experiments and for different purposes. In addition, we show that LEAF-E could also be used to fit datasets of other growth-related processes that follow the sigmoidal profile, such as cell length measurements along the leaf axis. Given its user-friendliness, ability to quantify duration and timing of leaf growth and broad application range, LEAF-E is a tool that could be routinely used to study growth processes following the sigmoidal profile

From lab to field : yield stability and shade avoidance genes are massively differentially expressed in the field

To unravel molecular mechanisms with the ultimate goal to achieve improved stress resilience or increased yield, plants are often studied under highly controlled conditions in which stresses are applied and in which growth‐ or architecture‐related traits are meticulously recorded. Over the past decades, this has led to a boost in our understanding of key molecular players and in strategies to improve yield stability. However, many single‐gene traits fail to translate into applications (Nuccio et al., 2018)

Correlation analysis of the transcriptome of growing leaves with mature leaf parameters in a maize RIL population

Background: To sustain the global requirements for food and renewable resources, unraveling the molecular networks underlying plant growth is becoming pivotal. Although several approaches to identify genes and networks involved in final organ size have been proven successful, our understanding remains fragmentary. Results: Here, we assessed variation in 103 lines of the Zea mays B73xH99 RIL population for a set of final leaf size and whole shoot traits at the seedling stage, complemented with measurements capturing growth dynamics, and cellular measurements. Most traits correlated well with the size of the division zone, implying that the molecular basis of final leaf size is already defined in dividing cells of growing leaves. Therefore, we searched for association between the transcriptional variation in dividing cells of the growing leaf and final leaf size and seedling biomass, allowing us to identify genes and processes correlated with the specific traits. A number of these genes have a known function in leaf development. Additionally, we illustrated that two independent mechanisms contribute to final leaf size, maximal growth rate and the duration of growth. Conclusions: Untangling complex traits such as leaf size by applying in-depth phenotyping allows us to define the relative contributions of the components and their mutual associations, facilitating dissection of the biological processes and regulatory networks underneath

RNA interference knockdown of BRASSINOSTEROID INSENSITIVE1 in maize reveals novel functions for brassinosteroid signaling in controlling plant architecture

Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbril complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbriI-RNAi transgenic lines have compromised BR signaling. zmbriI-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbriI-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbriI-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbriI-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling

An RNA in situ hybridization protocol optimized for monocot tissue

RNA in situ hybridization can be time-consuming and difficult to troubleshoot. Here, we provide an optimized protocol for maize leaf tissue, though it can be applied to other plant tissues such as shoot apical meristems, embryos, and floral organs. We generate three >100 bp unique antisense probes for each gene of interest and hybridize them to tissue sections

Tapping into the maize root microbiome to identify bacteria that promote growth under chilling conditions

Background When maize (Zea mays L.) is grown in the Northern hemisphere, its development is heavily arrested by chilling temperatures, especially at the juvenile phase. As some endophytes are beneficial for plants under stress conditions, we analyzed the impact of chilling temperatures on the root microbiome and examined whether microbiome-based analysis might help to identify bacterial strains that could promote growth under these temperatures. Results We investigated how the maize root microbiome composition changed by means of 16S rRNA gene amplicon sequencing when maize was grown at chilling temperatures in comparison to ambient temperatures by repeatedly cultivating maize in field soil. We identified 12 abundant and enriched bacterial families that colonize maize roots, consisting of bacteria recruited from the soil, whereas seed-derived endophytes were lowly represented. Chilling temperatures modified the root microbiome composition only slightly, but significantly. An enrichment of several chilling-responsive families was detected, of which the Comamonadaceae and the Pseudomonadaceae were the most abundant in the root endosphere of maize grown under chilling conditions, whereas only three were strongly depleted, among which the Streptomycetaceae. Additionally, a collection of bacterial strains isolated from maize roots was established and a selection was screened for growth-promoting effects on juvenile maize grown under chilling temperatures. Two promising strains that promoted maize growth under chilling conditions were identified that belonged to the root endophytic bacterial families, from which the relative abundance remained unchanged by variations in the growth temperature. Conclusions Our analyses indicate that chilling temperatures affect the bacterial community composition within the maize root endosphere. We further identified two bacterial strains that boost maize growth under chilling conditions. Their identity revealed that analyzing the chilling-responsive families did not help for their identification. As both strains belong to root endosphere enriched families, visualizing and comparing the bacterial diversity in these communities might still help to identify new PGPR strains. Additionally, a strain does not necessarely need to belong to a high abundant family in the root endosphere to provoke a growth-promoting effect in chilling conditions

Growth rate rather than growth duration drives growth heterosis in maize B104 hybrids

Research in maize is often performed using inbred lines that can be readily transformed, such as B104. However, because the B104 line flowers late, the kernels do not always mature before the end of the growing season, hampering routine seed yield evaluations of biotech traits introduced in B104 at many geographical locations. Therefore, we generated five hybrids by crossing B104 with the early-flowering inbred lines CML91, F7, H99, Mo17, and W153R and showed in three consecutive years that the hybrid lines proved to be suitable to evaluate seed yield under field conditions in a temperate climate. By assessing the two main processes driving maize leaf growth, being rate of growth (leaf elongation rate or LER) and the duration of growth (leaf elongation duration or LED) in this panel of hybrids, we showed that leaf growth heterosis was mainly the result of increased LER and not or to a lesser extent of LED. Ectopic expression of the transgenes GA20-oxidase (GA20-OX) and PLASTOCHRON1 (PLA1), known to stimulate the LER and LED, respectively, in the hybrids showed that leaf length heterosis can be stimulated by increased LER, but not by LED, indicating that LER rather than LED is the target for enhancing leaf growth heterosis. To enable transgenic maize research, hybrids between the inbred B104 that can be routinely transformed and early flowering inbreds were evaluated for yield components in three consecutive years. In addition, we show that leaf elongation rate is the main contributor to leaf growth heterosis in these hybrids, which can even be stimulated by overexpressing GA20OXIDASE, a known regulator of leaf elongation rate. Although leaf elongation duration has a limited contribution to the growth heterosis, the effect of the ectopic expression of PLASTOCHRON1, known to enhance leaf elongation duration and leaf growth, is still observed in the hybrids. This detailed understanding of the growth processes driving heterosis will be key to further breed for high yielding hybrids

How grass keeps growing : an integrated analysis of hormonal crosstalk in the maize leaf growth zone

We studied the maize leaf to understand how long-distance signals, auxin and cytokinin, control leaf growth dynamics. We constructed a mathematical model describing the transport of these hormones along the leaf growth zone and their interaction with the local gibberellin (GA) metabolism in the control of cell division. Assuming gradually declining auxin and cytokinin supply at the leaf base, the model generated spatiotemporal hormone distribution and growth patterns that matched experimental data. At the cellular level, the model predicted a basal leaf growth as a result of cell division driven by auxin and cytokinin. Superimposed on this, GA synthesis regulated growth through the control of the size of the region of active cell division. The predicted hormone and cell length distributions closely matched experimental data. To correctly predict the leaf growth profiles and final organ size of lines with reduced or elevated GA production, the model required a signal proportional to the size of the emerged part of the leaf that inhibited the basal leaf growth driven by auxin and cytokinin. Excision and shading of the emerged part of the growing leaf allowed us to demonstrate that this signal exists and depends on the perception of light intensity