Nathalie Gonzalez

Leaf size control: complex coordination of cell division and expansion

Size control of multicellular organisms poses a longstanding biological question that has always fascinated scientists. Currently the question is far from being resolved because of the complexity of and interconnection between cell division and cell expansion, two different events necessary to form a mature organ. Because of the importance of plants for food and renewable energy sources, dissecting the genetic networks underlying plant growth and organ size is becoming a high priority in plant science worldwide. Here, we review the current understanding of the cellular and molecular mechanisms that govern leaf organ size and discuss future prospects on research aiming at understanding organ size regulation.

Exit from proliferation during leaf development in **Arabidopsis thaliana** : a not-so-gradual process

Early leaf growth is sustained by cell proliferation and subsequent cell expansion that initiates at the leaf tip and proceeds in a basipetal direction. Using detailed kinematic and gene expression studies to map these stages during early development of the third leaf of Arabidopsis thaliana, we showed that the cell-cycle arrest front did not progress gradually down the leaf, but rather was established and abolished abruptly. Interestingly, leaf greening and stomatal patterning followed a similar basipetal pattern, but proliferative pavement cell and formative meristemoid divisions were uncoordinated in respect to onset and persistence. Genes differentially expressed during the transition from cell proliferation to expansion were enriched in genes involved in cell cycle, photosynthesis, and chloroplast retrograde signaling. Proliferating primordia treated with norflurazon, a chemical inhibitor of retrograde signaling, showed inhibited onset of cell expansion. Hence, differentiation of the photosynthetic machinery is important for regulating the exit from proliferation.

Survival and growth of Arabidopsis plants given limited water are not equal

Although drought tolerance is a central concern of plant research, the translatability for crop improvement is relatively low. Here we report on a major contributing factor to this lack of success. Drought tolerance is predominately scored based on an improved survival rate under lethal conditions that, as demonstrated by our study, does not predict superior growth performance and, thus, biomass yield gain, under moderate drought often encountered in the field.

Increased Leaf Size: Different Means to an End

The final size of plant organs, such as leaves, is tightly controlled by environmental and genetic factors that must spatially and temporally coordinate cell expansion and cell cycle activity. However, this regulation of organ growth is still poorly understood. The aim of this study is to gain more insight into the genetic control of leaf size in Arabidopsis (Arabidopsis thaliana) by performing a comparative analysis of transgenic lines that produce enlarged leaves under standardized environmental conditions. To this end, we selected five genes belonging to different functional classes that all positively affect leaf size when overexpressed: AVP1, GRF5, JAW, BRI1, and GA20OX1. We show that the increase in leaf area in these lines depended on leaf position and growth conditions and that all five lines affected leaf size differently; however, in all cases, an increase in cell number was, entirely or predominantly, responsible for the leaf size enlargement. By analyzing hormone levels, transcriptome, and metabolome, we provide deeper insight into the molecular basis of the growth phenotype for the individual lines. A comparative analysis between these data sets indicates that enhanced organ growth is governed by different, seemingly independent pathways. The analysis of transgenic lines simultaneously overexpressing two growth-enhancing genes further supports the concept that multiple pathways independently converge on organ size control in Arabidopsis.

The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion

The plant hormone auxin controls numerous aspects of plant growth and development by regulating the expression of hundreds of genes. SMALL AUXIN UP RNA (SAUR) genes comprise the largest family of auxin-responsive genes, but their function is unknown. Although prior studies have correlated the expression of some SAUR genes with auxin-mediated cell expansion, genetic evidence implicating SAURs in cell expansion has not been reported. The Arabidopsis SAUR19, SAUR20, SAUR21, SAUR22, SAUR23, and SAUR24 (SAUR19-24) genes encode a subgroup of closely related SAUR proteins. We demonstrate that these SAUR proteins are highly unstable in Arabidopsis. However, the addition of an N-terminal GFP or epitope tag dramatically increases the stability of SAUR proteins. Expression of these stabilized SAUR fusion proteins in Arabidopsis confers numerous auxin-related phenotypes indicative of increased and/or unregulated cell expansion, including increased hypocotyl and leaf size, defective apical hook maintenance, and altered tropic responses. Furthermore, seedlings expressing an artificial microRNA targeting multiple members of the SAUR19-24 subfamily exhibit short hypocotyls and reduced leaf size. Together, these findings demonstrate that SAUR19-24 function as positive effectors of cell expansion. This regulation may be achieved through the modulation of auxin transport, as SAUR gain-of-function and loss-of-function seedlings exhibit increased and reduced basipetal indole-3-acetic acid transport, respectively. Consistent with this possibility, SAUR19-24 proteins predominantly localize to the plasma membrane.

ANGUSTIFOLIA3 Binds to SWI/SNF Chromatin Remodeling Complexes to Regulate Transcription during Arabidopsis Leaf Development

The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell division during Arabidopsis leaf development. It is shown that AN3 associates with SWI/SNF chromatin remodeling complexes to regulate the expression of important downstream transcription factors and that the module SWI/SNF-AN3 is a major player in the transition from cell division to cell expansion in developing leaves. The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell proliferation during Arabidopsis thaliana leaf development, but the molecular mechanism is largely unknown. Here, we show that inducible nuclear localization of AN3 during initial leaf growth results in differential expression of important transcriptional regulators, including GROWTH REGULATING FACTORs (GRFs). Chromatin purification further revealed the presence of AN3 at the loci of GRF5, GRF6, CYTOKININ RESPONSE FACTOR2, CONSTANS-LIKE5 (COL5), HECATE1 (HEC1), and ARABIDOPSIS RESPONSE REGULATOR4 (ARR4). Tandem affinity purification of protein complexes using AN3 as bait identified plant SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling complexes formed around the ATPases BRAHMA (BRM) or SPLAYED. Moreover, SWI/SNF ASSOCIATED PROTEIN 73B (SWP73B) is recruited by AN3 to the promoters of GRF5, GRF3, COL5, and ARR4, and both SWP73B and BRM occupy the HEC1 promoter. Furthermore, we show that AN3 and BRM genetically interact. The data indicate that AN3 associates with chromatin remodelers to regulate transcription. In addition, modification of SWI3C expression levels increases leaf size, underlining the importance of chromatin dynamics for growth regulation. Our results place the SWI/SNF-AN3 module as a major player at the transition from cell proliferation to cell differentiation in a developing leaf.

David and Goliath: what can the tiny weed Arabidopsis teach us to improve biomass production in crops?

In the next decades, the world market for plant-derived products is expected to expand exponentially. Not only do we rely on plants to feed the growing world population, but plants will also play a pivotal role in providing a significant part of our increasing energy demands. Whereas in the 1960s the green revolution contributed to increase plant productivity, it is expected that biotechnological advances will further boost biomass production and plant yield. To do this effectively, it will be necessary to understand how the molecular machinery that determines yield parameters operates. Although of no direct economic significance, the model plant Arabidopsis can be used to find genes and regulatory networks controlling biomass production, which, in turn, can be applied for further growth improvement in other species including cereals.

Flower development schedule in tomato Lycopersicon esculentum cv. sweet cherry

The ontogeny of tomato (Lycopersicon esculentum cv. sweet cherry) flowers was subdivided into 20 stages using a series of landmark events. Stamen primordia emergence and carpel initiation occur at stage 4; archesporial and parietal tissue differentiate at stage 6 and meiosis in anthers begins at stage 9. Subepidermal meristematic ovule primordia are formed on the placenta at stage 9; megasporogenesis begins at stage 11–12 and embryo sac differentiation and ovule curvature take place at stage 14, once the pollen is maturing. We established a correlation between the characteristic cellular events in carpels and stamens and morphological markers of the perianth. The model of tomato flower development schedule was then used to analyse the spatial, temporal and tissue-specific expression of gene(s) involved in the regulation of floral organ development. As an example, the expression pattern of ORFX, a gene controlling cell size in tomato fruits, shows that expression starts very early during the ontogeny of reproductive organs.

Leaf Responses to Mild Drought Stress in Natural Variants of Arabidopsis

Arabidopsis accessions show different phenotypes in response to mild drought, yet a robust transcriptome response is conserved between the accessions. Although the response of plants exposed to severe drought stress has been studied extensively, little is known about how plants adapt their growth under mild drought stress conditions. Here, we analyzed the leaf and rosette growth response of six Arabidopsis (Arabidopsis thaliana) accessions originating from different geographic regions when exposed to mild drought stress. The automated phenotyping platform WIWAM was used to impose stress early during leaf development, when the third leaf emerges from the shoot apical meristem. Analysis of growth-related phenotypes showed differences in leaf development between the accessions. In all six accessions, mild drought stress reduced both leaf pavement cell area and number without affecting the stomatal index. Genome-wide transcriptome analysis (using RNA sequencing) of early developing leaf tissue identified 354 genes differentially expressed under mild drought stress in the six accessions. Our results indicate the existence of a robust response over different genetic backgrounds to mild drought stress in developing leaves. The processes involved in the overall mild drought stress response comprised abscisic acid signaling, proline metabolism, and cell wall adjustments. In addition to these known severe drought-related responses, 87 genes were found to be specific for the response of young developing leaves to mild drought stress.

The APC/C subunit 10 plays an essential role in cell proliferation during leaf development

The largest E3 ubiquitin-ligase complex, known as anaphase-promoting complex/cyclosome (APC/C), regulates the proteolysis of cell cycle regulators such as CYCLIN B and SECURIN that are essential for sister-chromatid separation and exit from mitosis. Despite its importance, the role of APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, the Arabidopsis thaliana APC/C subunit APC10 was characterized and shown to functionally complement an apc10 yeast mutant. The APC10 protein was located in specific nuclear bodies, most probably resulting from its association with the proteasome complex. An apc10 Arabidopsis knockout mutant strongly impaired female gametogenesis. Surprisingly, constitutive overexpression of APC10 enhanced leaf size. Through kinematic analysis, the increased leaf size was found to be due to enhanced rates of cell division during the early stages of leaf development and, at the molecular level, by increased APC/C activity as measured by an amplification of the proteolysis rate of the mitotic cyclin, CYCB1;1.