Osmotic stress inhibits leaf growth of Arabidopsis thaliana by enhancing ARF-mediated auxin responses.

We investigated the interaction between osmotic stress and auxin signaling in leaf growth regulation. Therefore, we grew Arabidopsis thaliana seedlings on agar media supplemented with mannitol to impose osmotic stress and 1-naphthaleneacetic acid (NAA), a synthetic auxin. We performed kinematic analysis and flow-cytometry to quantify the effects on cell division and expansion in the first leaf pair, determined the effects on auxin homeostasis and response (DR5::ß-glucuronidase), performed a next-generation sequencing transcriptome analysis and investigated the response of auxin-related mutants. Mannitol inhibited cell division and expansion. NAA increased the effect of mannitol on cell division, but ameliorated its effect on expansion. In proliferating cells, NAA and mannitol increased free IAA concentrations at the cost of conjugated IAA and stimulated DR5 promotor activity. Transcriptome analysis shows a large overlap between NAA and osmotic stress-induced changes, including upregulation of auxin synthesis, conjugation, transport and TRANSPORT INHIBITOR RESPONSE1 (TIR1) and AUXIN RESPONSE FACTOR (ARF) response genes, but downregulation of Aux/IAA response inhibitors. Consistently, arf7/19 double mutant lack the growth response to auxin and show a significantly reduced sensitivity to osmotic stress. Our results show that osmotic stress inhibits cell division during leaf growth of A. thaliana at least partly by inducing the auxin transcriptional response.

Control of leaf blade outgrowth and floral organ development by LEUNIG, ANGUSTIFOLIA3 and WOX transcriptional regulators.

Plant lateral organ development is a complex process involving both transcriptional activation and repression mechanisms. The WOX transcriptional repressor WOX1/STF, the LEUNIG (LUG) transcriptional corepressor and the ANGUSTIFOLIA3 (AN3) transcriptional coactivator play important roles in leaf blade outgrowth and flower development, but how these factors coordinate their activities remains unclear. Here we report physical and genetic interactions among these key regulators of leaf and flower development. We developed a novel in planta transcriptional activation/repression assay and suggest that LUG could function as a transcriptional coactivator during leaf blade development. MtLUG physically interacts with MtAN3, and this interaction appears to be required for leaf and flower development. A single amino acid substitution at position 61 in the SNH domain of MtAN3 protein abolishes its interaction with MtLUG, and its transactivation activity and biological function. Mutations in lug and an3 enhanced each other's mutant phenotypes. Both the lug and the an3 mutations enhanced the wox1 prs leaf and flower phenotypes in Arabidopsis. Our findings together suggest that transcriptional repression and activation mediated by the WOX, LUG and AN3 regulators function in concert to promote leaf and flower development, providing novel mechanistic insights into the complex regulation of plant lateral organ development.

Multiple mechanisms explain how reduced KRP expression increases leaf size of Arabidopsis thaliana.

Although cell number generally correlates with organ size, the role of cell cycle control in growth regulation is still largely unsolved. We studied kip related protein (krp) 4, 6 and 7 single, double and triple mutants of Arabidopsis thaliana to understand the role of cell cycle inhibitory proteins in leaf development. We performed leaf growth and seed size analysis, kinematic analysis, flow cytometery, transcriptome analysis and mathematical modeling of G1/S and G2/M checkpoint progression of the mitotic and endoreplication cycle. Double and triple mutants progressively increased mature leaf size, because of elevated expression of cell cycle and DNA replication genes stimulating progression through the division and endoreplication cycle. However, cell number was also already increased before leaf emergence, as a result of an increased cell number in the embryo. We show that increased embryo and seed size in krp4/6/7 results from seed abortion, presumably reducing resource competition, and that seed size differences contribute to the phenotype of several large-leaf mutants. Our results provide a new mechanistic understanding of the role of cell cycle regulation in leaf development and highlight the contribution of the embryo to the development of leaves after germination in general.

Functional Conservation in the SIAMESE-RELATED Family of Cyclin-Dependent Kinase Inhibitors in Land Plants.

The best-characterized members of the plant-specific SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors regulate the transition from the mitotic cell cycle to endoreplication, also known as endoreduplication, an altered version of the cell cycle in which DNA is replicated without cell division. Some other family members are implicated in cell cycle responses to biotic and abiotic stresses. However, the functions of most SMRs remain unknown, and the specific cyclin-dependent kinase complexes inhibited by SMRs are unclear. Here, we demonstrate that a diverse group of SMRs, including an SMR from the bryophyte Physcomitrella patens, can complement an Arabidopsis thaliana siamese (sim) mutant and that both Arabidopsis SIM and P. patens SMR can inhibit CDK activity in vitro. Furthermore, we show that Arabidopsis SIM can bind to and inhibit both CDKA;1 and CDKB1;1. Finally, we show that SMR2 acts to restrict cell proliferation during leaf growth in Arabidopsis and that SIM, SMR1/LGO, and SMR2 play overlapping roles in controlling the transition from cell division to endoreplication during leaf development. These results indicate that differences in SMR function in plant growth and development are primarily due to differences in transcriptional and posttranscriptional regulation, rather than to differences in fundamental biochemical function.

Three FLOWERING LOCUS T-like genes function as potential florigens and mediate photoperiod response in sorghum.

Sorghum is a typical short-day (SD) plant and its use in grain or biomass production in temperate regions depends on its flowering time control, but the underlying molecular mechanism of floral transition in sorghum is poorly understood. Here we characterized sorghum FLOWERING LOCUS T (SbFT) genes to establish a molecular road map for mechanistic understanding. Out of 19 PEBP genes, SbFT1, SbFT8 and SbFT10 were identified as potential candidates for encoding florigens using multiple approaches. Phylogenetic analysis revealed that SbFT1 clusters with the rice Hd3a subclade, while SbFT8 and SbFT10 cluster with the maize ZCN8 subclade. These three genes are expressed in the leaf at the floral transition initiation stage, expressed early in grain sorghum genotypes but late in sweet and forage sorghum genotypes, induced by SD treatment in photoperiod-sensitive genotypes, cooperatively repressed by the classical sorghum maturity loci, interact with sorghum 14-3-3 proteins and activate flowering in transgenic Arabidopsis plants, suggesting florigenic potential in sorghum. SD induction of these three genes in sensitive genotypes is fully reversed by 1 wk of long-day treatment, and yet, some aspects of the SD treatment may still make a small contribution to flowering in long days, indicating a complex photoperiod response mediated by SbFT genes.

Three-dimensional patterns of cell division and expansion throughout the development of Arabidopsis thaliana leaves.

Variations in size and shape of multicellular organs depend on spatio-temporal regulation of cell division and expansion. Here, cell division and expansion rates were quantified relative to the three spatial axes in the first leaf pair of Arabidopsis thaliana. The results show striking differences in expansion rates: the expansion rate in the petiole is higher than in the leaf blade; expansion rates in the lateral direction are higher than longitudinal rates between 5 and 10 days after stratification, but become equal at later stages of leaf blade development; and anticlinal expansion co-occurs with, but is an order of magnitude slower than periclinal expansion. Anticlinal expansion rates also differed greatly between tissues: the highest rates occurred in the spongy mesophyll and the lowest in the epidermis. Cell division rates were higher and continued for longer in the epidermis compared with the palisade mesophyll, causing a larger increase of palisade than epidermal cell area over the course of leaf development. The cellular dynamics underlying the effect of shading on petiole length and leaf thickness were then investigated. Low light reduced leaf expansion rates, which was partly compensated by increased duration of the growth phase. Inversely, shading enhanced expansion rates in the petiole, so that the blade to petiole ratio was reduced by 50%. Low light reduced leaf thickness by inhibiting anticlinal cell expansion rates. This effect on cell expansion was preceded by an effect on cell division, leading to one less layer of palisade cells. The two effects could be uncoupled by shifting plants to contrasting light conditions immediately after germination. This extended kinematic analysis maps the spatial and temporal heterogeneity of cell division and expansion, providing a framework for further research to understand the molecular regulatory mechanisms involved.

Freezing stress response of wild and cultivated chickpeas.

Chickpea is an economically and nutritionally important grain legume globally, however, cold stress has adverse effects on its growth. In cold countries, like Canada where the growing season is short, having cold stress-tolerant varieties is crucial. Crop wild relatives of chickpea, especially Cicer reticulatum, can survive in suboptimal environments and are an important resource for crop improvement. In this study, we explored the performance of eleven C. reticulatum wild accessions and two chickpea cultivars, CDC Leader and CDC Consul, together with a cold sensitive check ILC533 under freezing stress. Freezing tolerance was scored based on a 1-9 scale. The wild relatives, particularly Kesen_075 and CudiA_152, had higher frost tolerance compared to the cultivars, which all died after frost treatment. We completed transcriptome analysis via mRNA sequencing to assess changes in gene expression in response to freezing stress and identified 6,184 differentially expressed genes (DEGs) in CDC Consul, and 7,842 DEGs in Kesen_075. GO (gene ontology) analysis of the DEGs revealed that those related to stress responses, endogenous and external stimuli responses, secondary metabolite processes, and photosynthesis were significantly over-represented in CDC Consul, while genes related to endogenous stimulus responses and photosynthesis were significantly over-represented in Kesen_075. These results are consistent with Kesen_075 being more tolerant to freezing stress than CDC Consul. Moreover, our data revealed that the expression of CBF pathway-related genes was impacted during freezing conditions in Kesen_075, and expression of these genes is believed to alleviate the damage caused by freezing stress. We identified genomic regions associated with tolerance to freezing stress in an F2 population derived from a cross between CDC Consul and Kesen_075 using QTL-seq analysis. Eight QTLs (P<0.05) on chromosomes Ca3, Ca4, Ca6, Ca7, Ca8, and two QTLs (P<0.01) on chromosomes Ca4 and Ca8, were associated with tolerance to freezing stress. Interestingly, 58 DEGs co-located within these QTLs. To our knowledge, this is the first study to explore the transcriptome and QTLs associated with freezing tolerance in wild relatives of chickpea under controlled conditions. Altogether, these findings provide comprehensive information that aids in understanding the molecular mechanism of chickpea adaptation to freezing stress and further provides functional candidate genes that can assist in breeding of freezing-stress tolerant varieties.

Iron accumulation and partitioning in hydroponically grown wild and cultivated chickpea (Cicer arietinum L).

Chickpea (Cicer arietinum L.) is a staple food in many developing countries where iron (Fe) deficiency often occurs in their population. The crop is a good source of protein, vitamins, and micronutrients. Fe biofortification in chickpea can be part of long-term strategy to enhance Fe intake in human diet to help to alleviate Fe deficiency. To develop cultivars with high Fe concentration in seeds, understanding the mechanisms of absorption and translocation of Fe into the seeds is critical. An experiment was conducted using a hydroponic system to evaluate Fe accumulation in seeds and other organs at different growth stages of selected genotypes of cultivated and wild relatives of chickpea. Plants were grown in media with Fe zero and Fe added conditions. Six chickpea genotypes were grown and harvested at six different growth stages: V3, V10, R2, R5, R6, and RH for analysis of Fe concentration in roots, stems, leaves, and seeds. The relative expression of genes related to Fe-metabolism including FRO2, IRT1, NRAMP3, V1T1, YSL1, FER3, GCN2, and WEE1 was analyzed. The results showed that the highest and lowest accumulation of Fe throughout the plant growth stages were found in the roots and stems, respectively. Results of gene expression analysis confirmed that the FRO2 and IRT1 were involved in Fe uptake in chickpeas and expressed more in roots under Fe added condition. All transporter genes: NRAMP3, V1T1, YSL1 along with storage gene FER3 showed higher expression in leaves. In contrast, candidate gene WEE1 for Fe metabolism expressed more in roots under Fe affluent condition; however, GCN2 showed over-expression in roots under Fe zero condition. Current finding will contribute to better understanding of Fe translocation and metabolism in chickpea. This knowledge can further be used to develop chickpea varieties with high Fe in seeds.

Towards mechanistic models of plant organ growth.

Modelling and simulation are increasingly used as tools in the study of plant growth and developmental processes. By formulating experimentally obtained knowledge as a system of interacting mathematical equations, it becomes feasible for biologists to gain a mechanistic understanding of the complex behaviour of biological systems. In this review, the modelling tools that are currently available and the progress that has been made to model plant development, based on experimental knowledge, are described. In terms of implementation, it is argued that, for the modelling of plant organ growth, the cellular level should form the cornerstone. It integrates the output of molecular regulatory networks to two processes, cell division and cell expansion, that drive growth and development of the organ. In turn, these cellular processes are controlled at the molecular level by hormone signalling. Therefore, combining a cellular modelling framework with regulatory modules for the regulation of cell division, expansion, and hormone signalling could form the basis of a functional organ growth simulation model. The current state of progress towards this aim is that the regulation of the cell cycle and hormone transport have been modelled extensively and these modules could be integrated. However, much less progress has been made on the modelling of cell expansion, which urgently needs to be addressed. A limitation of the current generation models is that they are largely qualitative. The possibilities to characterize existing and future models more quantitatively will be discussed. Together with experimental methods to measure crucial model parameters, these modelling techniques provide a basis to develop a Systems Biology approach to gain a fundamental insight into the relationship between gene function and whole organ behaviour.

Genome-wide association analysis of stress tolerance indices in an interspecific population of chickpea.

Chickpea is a cool season crop that is highly vulnerable to abiotic stresses such as heat and drought. High temperature during early flowering and pod development stages significantly reduces the crop yield. The wild relatives of chickpeas can be potential donors for the introgression of heat and drought tolerance into cultivated chickpeas for crop improvement. Initially, 600 interspecific lines were derived from crosses between two elite cultivars, CDC Leader (kabuli chickpea) and CDC Consul (desi chickpea), and 20 accessions of Cicer reticulatum. The F5 interspecific lines were tested for agronomic and seed quality traits including reaction to ascochyta blight disease under field conditions at two locations in 2018. A subset of 195 lines were selected based on resistance to ascochyta blight and acceptable seed quality. These lines were evaluated for their performance under suboptimal conditions at Lucky Lake (2019 and 2020) and Moose Jaw (2019), Saskatchewan, Canada, and Yuma, Arizona, United States (2019-2020). The lines were grown and evaluated at two seeding dates, normal (SD1) and late (SD2) seeding dates, at each location and year. The same lines were genotyped using Cicer60K Axiom® SNP chip. The population structure was determined based on 35,431 informative SNPs using fastStructure, and the interspecific lines were clustered at a k-value of 15. Significant marker-trait associations were identified for seed yield from SD1 and SD2 seeding dates, and stress tolerance indices (ATI, K1STI, MP, SSPI, and TOL) using phenotypic values both from individual locations and combined analyses based on BLUP values. SNP marker Ca2_34600347 was significantly associated with yield from both the seeding dates. This and other SNP markers identified in this study may be useful for marker-assisted introgression of abiotic stress tolerance in chickpea.

A comprehensive technique for artificial hybridization in Chickpea (Cicer arietinum).

BACKGROUND: Two crossing techniques for hybridization of chickpea have been reported and include pollination after emasculation and pollination without emasculation. Success of crossing with emasculation varied from 5 to 17%; while the success rate varied from 20 to 50% by pollination without emasculation. The important reason for the low success rate of the two procedures could be lack of detailed information on the flowering stages chosen for crossing together with the environment where plants grow. RESULTS: We describe a comprehensive method for chickpea crossing where two genotypes, ICCV96029 as female and PI503023 as male parent were used. Leaf shape and seed size were used as morphological markers to select hybrids. For crossing, incision was made along the central line of the keel petal for the removal of anthers and to expose the stigma for placement of pollen from donor parent on its surface. After pollination, style was inserted back gently inside the keel petal and covered by wing petals and standard petals to make a natural sac which prevents drying of internal organs. Alternatively, if the conditions are favorable there is no need to protect the pollinated flower and therefore petal removal method for cross-pollination can be used. Our method showed around 78% crossing success rate which is much higher than the previous results. CONCLUSIONS: We have shown that the crossing by keel petal incision or petal removal is an effective approach which significantly increases the crossing success rate. Furthermore, our detailed method shows that the flowering stage, selection of parents and temperature play crucial roles in crossing success.

Leaf development: a cellular perspective.

Through its photosynthetic capacity the leaf provides the basis for growth of the whole plant. In order to improve crops for higher productivity and resistance for future climate scenarios, it is important to obtain a mechanistic understanding of leaf growth and development and the effect of genetic and environmental factors on the process. Cells are both the basic building blocks of the leaf and the regulatory units that integrate genetic and environmental information into the developmental program. Therefore, to fundamentally understand leaf development, one needs to be able to reconstruct the developmental pathway of individual cells (and their progeny) from the stem cell niche to their final position in the mature leaf. To build the basis for such understanding, we review current knowledge on the spatial and temporal regulation mechanisms operating on cells, contributing to the formation of a leaf. We focus on the molecular networks that control exit from stem cell fate, leaf initiation, polarity, cytoplasmic growth, cell division, endoreduplication, transition between division and expansion, expansion and differentiation and their regulation by intercellular signaling molecules, including plant hormones, sugars, peptides, proteins, and microRNAs. We discuss to what extent the knowledge available in the literature is suitable to be applied in systems biology approaches to model the process of leaf growth, in order to better understand and predict leaf growth starting with the model species Arabidopsis thaliana.