Do DEEPER ROOTING 1 Homologs Regulate the Lateral Root Slope Angle in Cucumber (Cucumis sativus)?

The architecture of the root system is fundamental to plant productivity. The rate of root growth, the density of lateral roots, and the spatial structure of lateral and adventitious roots determine the developmental plasticity of the root system in response to changes in environmental conditions. One of the genes involved in the regulation of the slope angle of lateral roots is DEEPER ROOTING 1 (DRO1). Its orthologs and paralogs have been identified in rice, Arabidopsis, and several other species. However, nothing is known about the formation of the slope angle of lateral roots in species with the initiation of lateral root primordia within the parental root meristem. To address this knowledge gap, we identified orthologs and paralogs of the DRO1 gene in cucumber (Cucumis sativus) using a phylogenetic analysis of IGT protein family members. Differences in the transcriptional response of CsDRO1, CsDRO1-LIKE1 (CsDRO1L1), and CsDRO1-LIKE2 (CsDRO1L2) to exogenous auxin were analyzed. The results showed that only CsDRO1L1 is auxin-responsive. An analysis of promoter-reporter fusions demonstrated that the CsDRO1, CsDRO1L1, and CsDRO1L2 genes were expressed in the meristem in cell files of the central cylinder, endodermis, and cortex; the three genes displayed different expression patterns in cucumber roots with only partial overlap. A knockout of individual CsDRO1, CsDRO1L1, and CsDRO1L2 genes was performed via CRISPR/Cas9 gene editing. Our study suggests that the knockout of individual genes does not affect the slope angle formation during lateral root primordia development in the cucumber parental root.

Root system architecture of historical spring wheat cultivars is associated with alleles and transcripts of major functional genes.

We evaluated root system architecture (RSA) of a set of 58 historical spring wheat cultivars from Pakistan representing 105 years of selection breeding. The evaluations were carried out under control and water-limited conditions using a high-throughput phenotyping system coupled with RhizoVision Explorer software. The cultivars were classified into three groups based on release year as cultivars released pre-1965, released between 1965 and 2000, and cultivars released post-2000. Under water-limited conditions a decline in 20 out of 25 RSA component traits was observed in pre-1965 cultivars group. Whereas cultivars released after the 1965, so-called green revolution period, showed a decline in 17 traits with significant increments in root length, depth, and steep angle frequency which are important root traits for resource-uptake under water-limited conditions. Similarly, cultivars released after 2000 indicated an increase in the number of roots, depth, diameter, surface area, and steep angle frequency. The coefficient of correlation analysis showed a positive correlation between root depth and yield-related traits under water-limited conditions. We also investigated the effects of green-revolution genes (Rht1) and some phenology-related genes such as DRO1, TaMOR, TaLTPs, TaSus-2B on RSA and identified significant associations of these genes with important root traits. There was strong selection pressure on DRO1 gene in cultivated wheat indicating the allele fixed in modern wheat cultivars is different from landraces. The expression of DRO1, and TaMOR were retrieved from an RNAseq experiment, and results were validated using qRT-PCR. The highest expression of DRO1 and TaMOR was found in Chakwal-50, a rainfed cultivar released in 2008, and MaxiPak-65 released in 1965. We conclude that there is a positive historic change in RSA after 1965 that might be attributed to genetic factors associated with favored RSA traits. Furthermore, we suggest root depth and steep angle as promising traits to withstand water-limited environments and may have implications in selection for breeding.

Challenges to design-oriented breeding of root system architecture adapted to climate change.

Roots are essential organs for capturing water and nutrients from the soil. In particular, root system architecture (RSA) determines the extent of the region of the soil where water and nutrients can be gathered. As global climate change accelerates, it will be important to improve belowground plant parts, as well as aboveground ones, because roots are front-line organs in the response to abiotic stresses such as drought, flooding, and salinity stress. However, using conventional breeding based on phenotypic selection, it is difficult to select breeding lines possessing promising RSAs to adapted to abiotic stress because roots remain hidden underground. Therefore, new breeding strategies that do not require phenotypic selection are necessary. Recent advances in molecular biology and biotechnology can be applied to the design-oriented breeding of RSA without phenotypic selection. Here I summarize recent progress in RSA ideotypes as "design" and RSA-related gene resources as "materials" that will be needed in leveraging these technologies for the RSA breeding. I also highlight the future challenges to design-oriented breeding of RSA and explore solutions to these challenges.

Allele mining for a drought responsive gene DRO1 determining root growth angle in donors of drought tolerance in rice (Oryza sativa L.).

Deeper Rooting 1 (DRO1) gene identified from a major QTL on chromosome 9 increases the root growth angle (RGA) and thus facilitates survival under drought and hence is an excellent candidate for rice improvement. Twenty-four major Indian upland and lowland genotypes including the 'yield under drought' (DTY) QTL donors were subjected to allele mining of DRO1 (3058 bp) using four pairs of overlapping primers. A total of 216 and 52 SNPs were identified across all genotypes in the gene and coding region (756 bp) respectively with transversions 3.6 fold more common than transitions in the gene and 2.5 times in the CDS. In 251 amino acid long protein, substitutions were found in 19 positions, wherein change in position 92 was the most frequent. Based on allele mining, the 24 genotypes can be classified into 16 primary structure variants ranging from complete functional allele (Satti, IR36 and DTY 3.1 donor, IR81896-B-B-195) to truncated non-functional alleles in PMK2, IR64, IR20 and Swarna. All the DTY donors, other than IR81896-B-B-195, and most of the upland drought tolerant cultivars (Nagina 22, Vandana and Dhagaddeshi) had accumulated 6-19 SNPs and 4-8 amino acid substitutions resulting in substantial differences in their protein structure. The expression analysis revealed that all the genotypes showed upregulation under drought stress though the degree of upregulation varied among genotypes. The information on structural variations in DRO1 gene will be very useful for the breeders, especially in the light of recent breeding programmes on improving drought tolerance using several DTY donors and upland accessions. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s12298-021-00950-2).

The intersection of nitrogen nutrition and water use in plants: new paths toward improved crop productivity.

Water and nitrogen availability limit crop productivity globally more than most other environmental factors. Plant availability of macronutrients such as nitrate is, to a large extent, regulated by the amount of water available in the soil, and, during drought episodes, crops can become simultaneously water and nitrogen limited. In this review, we explore the intricate relationship between water and nitrogen transport in plants, from transpiration-driven mass flow in the soil to uptake by roots via membrane transporters and channels and transport to aerial organs. We discuss the roles of root architecture and of suberized hydrophobic root barriers governing apoplastic water and nitrogen movement into the vascular system. We also highlight the need to identify the signalling cascades regulating water and nitrogen transport, as well as the need for targeted physiological analyses of plant traits influencing water and nitrogen uptake. We further advocate for incorporation of new phenotyping technologies, breeding strategies, and agronomic practices to improve crop yield in water- and nitrogen-limited production systems.

Isolation and Molecular Characterisation of TtDro1A and TtDro1B Genes from Triticum turgidum Subspecies durum and turgidum, Study of Their Influences on Seedling Root Angles.

Durum wheat (Triticum turgidum, 2n = 4x = AABB) includes several subspecies with differential characteristics in their root system architecture (RSA). Subspecies durum has longer and more vertical roots, while subspecies turgidum has smaller and shallower roots. The homeologous genes TtDro1A and TtDro1B of both subspecies have been identified and found to differ in their sizes, sequences and the proteins they encode. To determine whether there is a relationship between the level of expression of these two genes and the angle adopted by the roots of durum wheat seedlings, their expressions has been studied by RT-qPCR, both in the primary seminal root and in the other seminal roots. The results of the analyses showed that the TtDro1A gene is expressed 1.4 times more in the primary seminal root than in the other seminal roots. Furthermore, this gene is expressed 2.49 to 8.76 times more than TtDro1B depending on root type (primary or seminal) and subspecies. There are positive correlations between the expression ratio of both genes (TtDro1A/TtDro1B) and the mean of all root angles, the most vertical root angle and the most horizontal root angle of the seedlings. The higher the expression of TtDro1B gene, the lower the root growth angles.

Characteristics of members of IGT family genes in controlling rice root system architecture and tiller development.

Root system architecture (RSA) and tiller are important agronomic traits. However, the mechanisms of the IGT family genes regulate RSA and tiller development in different rice varieties remain unclear. In this study, we demonstrated that 38 rice varieties obtained from Yuanyang Hani's terraced fields with different RSA and could be classified into six groups based on the ratio of root length and width. We found a positive correlation between RSA (including root width, length, and area) and tiller number in most of rice varieties. Furthermore, the IGT family genes Deeper Rooting 1 (DRO1), LAZY1, TAC1, and qSOR1 showed different expression patterns when rice grown under irrigation and drought conditions. Moreover, the qSOR1 gene had higher levels in the roots and tillers, and accompanied with higher levels of PIN1b gene in roots when rice grown under drought environmental condition. DRO1 gene had two single nucleotide polymorphisms (SNPs) in the exon 3 sequences and showed different expression patterns in the roots and tillers of the 38 rice varieties. Overexpression of DRO1 with a deletion of exon 5 caused shorter root length, less lateral roots and lower levels of LAZY1, TAC1, and qSOR1. Further protein interaction network, microRNA targeting and co-expression analysis showed that DRO1 plays a critical role in the root and tiller development associated with auxin transport. These data suggest that the RSA and tiller development are regulated by the IGT family genes in an intricate network way, which is tightly related to rice genetic background in rice adapting to different environmental conditions.

Loss of DRO1/CCDC80 in the tumor microenvironment promotes carcinogenesis.

Tumors are composed of the tumor cells and the surrounding microenvironment. Both are closely interwoven and interact by a complex and multifaceted cross-talk which plays an integral part in tumor initiation, growth, and progression. Dro1/Ccdc80 has been shown to be a potent suppressor of colorectal cancer and ubiquitous inactivation of Dro1/Ccdc80 strongly promoted colorectal carcinogenesis in ApcMin/+ mice and in a chemically-induced colorectal cancer model. The aim of the present study was to investigate whether Dro1/Ccdc80's tumor suppressive function is tumor-cell-autonomous. Expression of Dro1/Ccdc80 in cancer cells had no effect on both colon tumor development in ApcMin/+ mice and formation of xenograft tumors. In contrast, DRO1/CCDC80 loss in the microenvironment strongly increased tumor growth in xenograft models, inhibited cancer cell apoptosis, and promoted intestinal epithelial cell migration. Moreover, stromal Dro1/Ccdc80 inactivation facilitated formation of intestinal epithelial organoids. Expression analyses showed Dro1/Ccdc80 to be significantly down-regulated in murine gastric cancer associated fibroblasts, in ApcMin/+ colon tumor primary stromal cells and in microdissected stroma from human colorectal cancer compared to normal, non-tumor stroma. Our results demonstrate epithelial derived DRO1/CCDC80 to be dispensable for intestinal tissue homeostasis and identify Dro1/Ccdc80 as tumor suppressor in the tumor microenvironment.

Loss of DRO1/CCDC80 results in obesity and promotes adipocyte differentiation.

To investigate the role of DRO1 in obesity and adipogenesis in vivo, we generated a constitutive Dro1 knockout mouse model and analyzed the effect of DRO1 loss on body growth under standard and high fat diet feeding conditions. Loss of DRO1 resulted in a significant increase in body weight which was accompanied by a substantial expansion of white adipose tissue depots. The obese phenotype could be further enhanced by a high fat dietary challenge which also resulted in impaired glucose metabolism and the development of hepatosteatosis in Dro1 knockout mice. To study the role of DRO1 in adipocyte differentiation, primary stromal-vascular (SV) cells were isolated from inguinal white fat pads of knockout and control mice. In primary SV cells, depletion of DRO1 significantly promoted adipogenesis with upregulation of markers for adipogenesis (Cebpa, Pparg, Adipoq) and lipid metabolism (Dgat1, Dgat2). Our results demonstrate that DRO1 is a crucial regulator of energy homeostasis in vivo and functions as an inhibitor of adipogenesis in primary cells.

QTLs underlying natural variation of root growth angle among rice cultivars with the same functional allele of DEEPER ROOTING 1.

BACKGROUND: The functional allele of the rice gene DEEPER ROOTING 1 (DRO1) increases the root growth angle (RGA). However, wide natural variation in RGA is observed among rice cultivars with the functional DRO1 allele. To elucidate genetic factors related to such variation, we quantitatively measured RGA using the basket method and analyzed quantitative trait loci (QTLs) for RGA in three F2 mapping populations derived from crosses between the large RGA-type cultivar Kinandang Patong and each of three accessions with varying RGA: Momiroman has small RGA and was used to produce the MoK-F2 population; Yumeaoba has intermediate RGA (YuK-F2 population); Tachisugata has large RGA (TaK-F2 population). All four accessions belong to the same haplotype group of functional DRO1 allele. RESULTS: We detected the following statistically significant QTLs: one QTL on chromosome 4 in MoK-F2, three QTLs on chromosomes 2, 4, and 6 in YuK-F2, and one QTL on chromosome 2 in TaK-F2. Among them, the two QTLs on chromosome 4 were located near DRO2, which has been previously reported as a major QTL for RGA, whereas the two major QTLs for RGA on chromosomes 2 (DRO4) and 6 (DRO5) were novel. With the LOD threshold reduced to 3.0, several minor QTLs for RGA were also detected in each population. CONCLUSION: Natural variation in RGA in rice cultivars carrying functional DRO1 alleles may be controlled by a few major QTLs and by several additional minor QTLs.

A QTL for root growth angle on rice chromosome 7 is involved in the genetic pathway of DEEPER ROOTING 1.

BACKGROUND: Root growth angle (RGA) is an important trait that influences the ability of rice to avoid drought stress. DEEPER ROOTING 1 (DRO1), which is a major quantitative trait locus (QTL) for RGA, is responsible for the difference in RGA between the shallow-rooting cultivar IR64 and the deep-rooting cultivar Kinandang Patong. However, the RGA differences between these cultivars cannot be fully explained by DRO1. The objective of this study was to identify new QTLs for RGA explaining the difference in RGA between these cultivars. RESULTS: By crossing IR64 (which has a non-functional allele of DRO1) with Kinandang Patong (which has a functional allele of DRO1), we developed 26 chromosome segment substitution lines (CSSLs) that carried a particular chromosome segment from Kinandang Patong in the IR64 genetic background. Using these CSSLs, we found only one chromosomal region that was related to RGA: on chromosome 9, which includes DRO1. Using an F2 population derived from a cross between Kinandang Patong and the Dro1-NIL (near isogenic line), which had a functional DRO1 allele in the IR64 genetic background, we identified a new QTL for RGA (DRO3) on the long arm of chromosome 7. CONCLUSIONS: DRO3 may only affect RGA in plants with a functional DRO1 allele, suggesting that DRO3 is involved in the DRO1 genetic pathway.

The cl2/dro1/ccdc80 null mice develop thyroid and ovarian neoplasias.

We have previously reported that the expression of the CL2/CCDC80 gene is downregulated in human papillary thyroid carcinomas, particularly in follicular variants. We have also reported that the restoration of CL2/CCDC80 expression reverted the malignant phenotype of thyroid carcinoma cell lines and that CL2/CCDC80 positively regulated E-cadherin expression, an ability that likely accounts for the role of the CL2/CCDC80 gene in thyroid cancer progression. In order to validate the tumour suppressor role of the CL2/CCDC80 gene in thyroid carcinogenesis we generated cl2/ccdc80 knock-out mice. We found that embryonic fibroblasts from cl2/ccdc80(-/-) mice showed higher proliferation rate and lower susceptibility to apoptosis. Furthermore, cl2/ccdc80(-/-) mice developed thyroid adenomas and ovarian carcinomas. Finally, ret/PTC1 transgenic mice crossed with the cl2/ccdc80 knock-out mice developed more aggressive thyroid carcinomas compared with those observed in the single ret/PTC1 transgenic mice. Together, these results indicate CL2/CCDC80 as a putative tumour suppressor gene in human thyroid carcinogenesis.