African Oryza glaberrima and Oryza sativa landraces are considered valuable resources for breeding traits due to their adaptation to local environmental and soil conditions. They often possess ...superior resistance to endemic pests and tolerance to drought and nutrient deficiencies when compared to the "imported" high production Asian rice varieties. In contrast, "domestication traits" such as seed shattering, lodging, and seed yield are not well established in these African landraces. Therefore, the use of these African varieties for high production agriculture is limited by unpredictable yield and grain quality. We are addressing this shortcoming by developing protocols for genetically transforming African landraces to allow the use of CRISPR-Cas mediated breeding approaches. Here we use as proof of concept the cultivated African landrace Kabre to target selected known "domestication loci" and improve the agronomic potential of Kabre rice. Stable genetic transformation with CRISPR-Cas9-based vectors generated single and simultaneous multiple gene knockouts. Plants with reduced stature to diminish lodging were generated by disrupting the HTD1 gene. Furthermore, three loci shown to control seed size and/or yield (GS3, GW2 and GN1A) were targeted using a multiplex CRISPR-Cas9 construct. This resulted in mutants with significantly improved seed yield. Our study provides an example of how new breeding technologies can accelerate the development of highly productive African landrace rice varieties, an important advancement considering that Africa is a hotspot for worldwide population growth and therefore prone to food shortage.
Alternative splicing (AS) is a key modulator of development in many eukaryotic organisms. In plants, alternative splice forms of non-coding RNAs (ncRNAs) are known to modulate flowering time in ...Arabidopsis and fertility in rice. Here we demonstrate that alternative splicing of coding and long non-coding RNAs occurs during rice seed development by comparing AS in immature seeds vs. embryo and endosperm of mature seeds. Based on computational predictions of AS events determined from a Bayesian analysis of junction counts of RNA-seq datasets, differential splicing of protein-coding, and non-coding RNAs was determined. In contrast to roots, leaves, flowers, buds, and reproductive meristems, developing seeds had 5.8-57 times more predicted AS. Primers designed to span introns and exons were used to detect AS events predicted by rMATs in cDNA derived from early (milk) seed, embryo, and endosperm. Comparing milk seed vs. mature embryo and endosperm, AS of
(a gene implicated in epigenetic gene silencing), was markedly different. Long non-coding RNAs (lncRNAs) also underwent AS during the transition from milk seed to mature embryo and endosperm, with a complex gene structure, and were more extensively processed than predicted by current genome annotation. Exon retention of lncRNAs was enhanced in embryos. Searching all 5,515 lncRNAs in the NCBI genome annotation uncovered gene families based on highly conserved regions shared by groups of 3-35 lncRNAs. The homologies to other lncRNAs, as well as homologies to coding sequences, and the genomic context of lncRNAs provide inroads for functional analysis of multi-exonic lncRNAs that can be extensively processed during seed development.
Summary
Little is known about the signalling processes involved in the response of roots to abiotic stresses. The Arabidopsis root is a model system of root anatomy with a simple architecture and is ...amenable to genetic manipulation. Although it is known that the root responds to cold, drought and salt stress with increases in cytoplasmic free calcium, there is currently no information about the role(s) of the functionally diverse cell types that comprise the root. Transgenic Arabidopsis with enhancer‐trapped GAL4 expression in specific cell types was used to target the calcium reporting protein, aequorin, fused to a modified yellow fluorescent protein (YFP). The luminescence output of targeted aequorin enabled in vivo measurement of changes in cytosolic free calcium concentrations (Ca2+cyt) in specific cell types during acute cold, osmotic and salt stresses. In response to an acute cold stress, all cell types tested as well as plants constitutively expressing aequorin displayed rapid Ca2+cyt peaks. However, there were significant quantitative differences between different cell types in terms of their response to cold stress, osmotic stress (440 mM mannitol) and salt stress (220 mM NaCl), implying specific roles for certain cell types in the detection and/or response to these stimuli. In response to osmotic and salt stress, the endodermis and pericycle displayed prolonged oscillations in cytosolic calcium that were distinct from the responses of the other cell types tested. Targeted expression of aequorin circumvented the technical difficulties involved in fluorescent dye injection as well as the lack of cell specificity of constitutively expressed aequorin, and revealed a new level of complexity in root calcium signalling.
Seeds are one of the most important food sources, providing humans and animals with essential nutrients. These nutrients include carbohydrates, lipids, proteins, vitamins and minerals. Carbohydrates ...are one of the main energy sources for both plant and animal cells and play a fundamental role in seed development, human nutrition and the food industry. Many studies have focused on the molecular pathways that control carbohydrate flow during seed development in monocot and dicot species. For this reason, an overview of seed biodiversity focused on the multiple metabolic and physiological mechanisms that govern seed carbohydrate storage function in the plant kingdom is required. A large number of mutants affecting carbohydrate metabolism, which display defective seed development, are currently available for many plant species. The physiological, biochemical and biomolecular study of such mutants has led researchers to understand better how metabolism of carbohydrates works in plants and the critical role that these carbohydrates, and especially starch, play during seed development. In this review, we summarize and analyze the newest findings related to carbohydrate metabolism’s effects on seed development, pointing out key regulatory genes and enzymes that influence seed sugar import and metabolism. Our review also aims to provide guidelines for future research in the field and in this way to assist seed quality optimization by targeted genetic engineering and classical breeding programs.
Cell wall modifications are of pivotal importance during plant development. Among cell wall components, xyloglucans are the major hemicellulose polysaccharide in primary cell walls of dicots and ...non-graminaceous monocots. They can connect the cellulose microfibril surface to affect cell wall mechanical properties. Changes in xyloglucan structure are known to play an important role in regulating cell growth. Therefore, the degradation of xyloglucan is an important modification that alters the cell wall. The α-XYLOSIDASE1 (XYL1) gene encodes the only α-xylosidase acting on xyloglucans in Arabidopsis thaliana. Here, we showed that mutation of XYL1 strongly influences seed size, seed germination, and fruit elongation. We found that the expression of XYL1 is directly regulated in developing seeds and fruit by the MADS-box transcription factor SEEDSTICK. We demonstrated that XYL1 complements the stk smaller seed phenotype. Finally, by atomic force microscopy, we investigated the role of XYL1 activity in maintaining cell stiffness and growth, confirming the importance of cell wall modulation in shaping organs.
In salt-tolerant Chara longifolia, enhanced Na+ efflux plays an important role in maintaining low cytoplasmic Na+. When it is cultured in fresh water (FW), C. longifolia has a higher Na+ efflux than ...the obligate FW Chara corallina, although pH dependence and inhibitor profiles are similar for both species (J. Whittington and M.A. Bisson 1994 J Exp Bot 45: 657-665). When it is cultured in saltwater, C. longifolia has a Na+ efflux of 264 +/- 14 nmol m-2 s-1 at pH 7, 13 times higher than FW-adapted cultures and 31 times higher than C. corallina. As in FW-adapted plants, efflux is highest at pH 5, but pH dependence is less steep and more linear in cells adapted to saltwater. In plants of both species from FW cultures, Na+ efflux is inhibited by Li+ at pH 5 but not at pH 7 or 9, whereas in the salt-adapted C. longifolia, Li+ inhibits Na+ efflux at pH 7 and 9 but not at pH 5. Amiloride inhibits Na+ efflux in salt-adapted cells but not in FW cells. We conclude that a new type of Na+ efflux system is induced in salt-adapted plants, although both systems have characteristics suggestive of a Na+/H+ antiport. In all cases, a 1:1 Na+/H+ antiport would have sufficient energy to maintain the cytoplasmic Na+ activities measured at pH 5 and 7 but not at pH 9, which suggests that another efflux system must be operating at pH 9.
Salt-tolerant plants must be able to maintain low cytoplasmic Na$\sp+$ levels. In salt-tolerant Chara longifolia, enhanced Na$\sp+$ efflux plays an important role. When cultured in salt water, C. ...longifolia has a Na$\sp+$ efflux of 264 $\pm$ 14 nmol m$\sp{-2}$ s$\sp{-1}$ at pH 7, 13 times higher than the rate of freshwater-adapted cultures and 31 times higher than the rate of freshwater-obligate Chara corallina. As in freshwaeer-adapted plants, efflux is highest at pH 5, but pH dependence is less steep and more linear in cells adapted to salt water. The response to inhibitors is also different. In plants of both species from freshwater cultures, Na$\sp+$ efflux is inhibited by Li$\sp+$ at pH 5 but not 7 or 9, whereas in the salt-adapted C. longifolia, Li$\sp+$ inhibits at pH 7 and 9 but not at pH 5. Amiloride inhibits efflux in the salt-adapted cells but not in cells from freshwater culture. We therefore conclude that a new type of Na$\sp+$ efflux system is induced under salt stress, although both systems have characteristics suggestive of a Na$\sp+$/H$\sp+$ antiport. Efflux of Na$\sp+$ from C. longifolia may be due to the activity of a Na$\sp+$/H$\sp+$ antiport driven by the primary active transport of the P-type H$\sp+$-ATPase, and/or by the activity of a Na$\sp+$-ATPase. We attempted to isolate P-type ATPases from C. longifolia using a PCR-based cloning strategy, and obtained two clones. The first had homology to prokaryotic cation-transporting ATPases and eukaryotic Na$\sp+$/K$\sp+$ ATPases. The second showed high homology to the B subunit of the E. coli high affinity K$\sp+$-ATPase kdp. Taken together, these studies suggest that molecular cloning and expression analysis using Chara is feasible and may reveal an interesting suite of ion transport mechanisms at the molecular genetic level.
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