Experiments were designed to compare the relationship between starch degradation and the use of carbon for maintenance and growth in Arabidopsis in source‐limited and sink‐limited conditions. It is ...known that starch degradation is regulated by the clock in source‐limited plants, which degrade their starch in a linear manner such that it is almost but not completely exhausted at dawn. We asked whether this response is maintained under an extreme carbon deficit. Arabidopsis was subjected to a sudden combination of a day of low irradiance, to decrease starch at dusk, and a warm night. Starch was degraded in a linear manner through the night, even though the plants became acutely carbon starved. We conclude that starch degradation is not increased to meet demand in carbon‐limited plants. This network property will allow stringent control of starch turnover in a fluctuating environment. In contrast, in sink‐limited plants, which do not completely mobilize their starch during the night, starch degradation was accelerated in warm nights to meet the increased demand for maintenance and growth. Across all conditions, the rate of growth at night depends on the rate of starch degradation, whereas the rate of maintenance respiration decreases only when starch degradation is very slow.
Summary
Kiwifruit (Actinidia chinensis) is a dioecious, long‐living woody perennial vine. Reduced generation time and induction of hermaphroditism can accelerate crop improvement and facilitate ...alternative farming for better food security in the face of climate change.
Previous studies identified that CENTRORADIALIS genes CEN and CEN4 act to repress flowering, whilst the male‐specific Shy Girl (SyGl) gene with homology to type‐C cytokinin response regulators could repress gynoecium development in model plants. Here we use CRISPR/Cas9 to mutagenize CEN, CEN4 and SyGl in the male kiwifruit A. chinensis ‘Bruce’.
Biallelic mutations of CEN and CEN4 generated rapid‐flowering male plants, and simultaneous targeting of CEN4 and SyGl gave rise to rapid‐flowering hermaphrodites with restored gynoecial function and viable pollen, providing functional evidence for the role of SyGl in suppression of feminization. Analysis of ovary tissues identified genes that contribute to carpel development and revealed that SyGl affected both cytokinin profiles and the expression of genes involved in cytokinin metabolism and signalling. The plant lines generated by CEN4/SyGl knockout could self‐pollinate and produce fast‐flowering offspring.
These results establish that SyGI acts as the suppressor of feminization in kiwifruit and demonstrate the potential for accelerated breeding in an outcrossing horticultural woody perennial.
Pollen development, from unicellular microspores to anthesis, is a complex process involving the coordinated specification, differentiation and functions of different cell types. Key to understanding ...this development is identifying the genes expressed at precise stages of development. However, transcriptomic studies on pollen prior to anthesis are complicated by the inaccessible nature of pollen developing in the anther and the resistant pollen wall. To assist with understanding gene expression during pollen development we have developed a protocol to perform RNA-Seq on pollen isolated from a single anther (SA RNA-Seq). The protocol involves removing pollen from a single anther for analysis and viewing the remaining pollen to determine the developmental stage. The isolated pollen is chemically lysed and mRNA isolated from the lysate using an oligo-dT column before library preparation. Here, we report on the development and testing of our method and the generation of a transcriptome for three stages of pollen development from Arabidopsis (
) and two stages from male kiwifruit (
). This protocol enables the transcriptome of precise developmental stages of pollen to be analyzed, and uses a small number of plants, potentially facilitating studies that require a range of treatments or the analysis of the first generation of transgenic plants.
Dioecy, the presence of male and female individuals, has evolved independently in multiple flowering plant lineages
. Although theoretical models for the evolution of dioecy, such as the ...'two-mutations' model, are well established
, little is known about the specific genes determining sex and their evolutionary history
. Kiwifruit, a major tree crop consumed worldwide, is a dioecious species. In kiwifruit we previously identified a Y-encoded sex-determinant candidate gene acting as the suppressor of feminization (SuF), named Shy Girl (SyGI)
. Here, we identify a second Y-encoded sex-determinant that we named Friendly Boy (FrBy), which exhibits strong expression in tapetal cells. Gene-editing and complementation analyses in Arabidopsis thaliana and Nicotiana tabacum indicated that FrBy acts for the maintenance of male (M) functions, independently of SyGI, and that these functions are conserved across angiosperm species. We further characterized the genomic architecture of the small (<1 megabase pairs (Mb)) male-specific region of the Y chromosome (MSY), which harbours only two genes expressed extensively in developing gynoecia and androecia, respectively: SyGI and FrBy. Re-sequencing of the genome of a natural hermaphrodite kiwifruit revealed that this individual is genetically male but carries deletion(s) of parts of the Y chromosome, including SyGI. Additionally, expression of FrBy in female kiwifruit resulted in hermaphrodite plants. These results clearly indicate that Y-encoded SyGI and FrBy act independently as the SuF and M factors in kiwifruit, respectively, and provide insight into not only the evolutionary path leading to a two-factor sex-determination system, but also a new breeding approach for dioecious species.
Chlorophyll is present in many plant organs, including immature fruit where it is usually degraded during ripening. Mature green kiwifruit (Actinidia deliciosa) are an exception, with high ...concentrations of chlorophyll remaining in the fruit flesh. In gold-fleshed kiwifruit (A. chinensis), chlorophyll is degraded to colourless catabolites upon fruit ripening, leaving yellow carotenoids visible. We have identified candidate genes for the control of chlorophyll degradation in kiwifruit and examined the transcript levels of these genes in maturing kiwifruit using quantitative real-time PCR. Results indicate that the biosynthesis and degradation, or turnover, of chlorophyll is transcriptionally regulated in green-and gold-fleshed kiwifruit. Both species of kiwifruit were found to have two homologues of the stay-green gene (SGR), a small protein that is postulated to aid in the dismantling of the light- harvesting complex, allowing free chlorophyll to enter the degradation pathway. However, with the exception of very mature green fruit, where degreening was observed, SGR2 was more highly expressed in gold fruit, indicating a potential regulatory step of chlorophyll degradation. When the SGR genes were over-expressed in tobacco leaves, degreening was observed. Our results show that chlorophyll degradation is differentially regulated in kiwifruit, and suggest that gold kiwifruit transcribe more degradation genes, leading to earlier and more sustained chlorophyll degradation in this fruit than in green kiwifruit.
Key message
We describe a simple method to view meiotic cells in whole anthers from a range of plants. The method retains spatial organisation and enables simultaneous analysis of many meiotic cells.
...Understanding the process of male meiosis in flowering plants, and the role of genes involved in this process, offers potential for plant breeding, such as through increasing the level of genetic variation or the manipulation of ploidy levels in the gametes. A key to the characterisation of meiotic gene function and meiosis in non-model crop plants, is the analysis of cells undergoing meiosis, a task made difficult by the inaccessible nature of these cells. Here, we describe a simple and rapid method to analyse plant male meiosis in intact anthers in a range of plant species. This method allows analysis of numerous cells undergoing meiosis and, as meiotic cells stay within the anther, it retains information of the three-dimensional organisation and the location of organelles in meiotic cells. We show that the technique provides information on male meiosis by looking at the synchrony of meiotic progression between and within locules, and comparing wildtype and mutant plants through the chromosome separation stages in
Arabidopsis thaliana
. Additionally, we demonstrate that the protocol can be adopted to other plants with different floral morphology using
Medicago truncatula
as an example with small floral buds and the non-model plant kiwifruit (
Actinidia chinensis
) with larger buds and anthers.
• Background and Aims Green kiwifruit (Actinidia deliciosa) retain high concentrations of chlorophyll in the fruit flesh, whereas in gold-fleshed kiwifruit (A. chinensis) chlorophyll is degraded to ...colourless catabolites during fruit development, leaving yellow carotenoids visible. The plant hormone group the cytokinins has been implicated in the delay of senescence, and so the aim of this work was to investigate the link between cytokinin levels in ripening fruit and chlorophyll de-greening. • Methods The expression of genes related to cytokinin metabolism and signal transduction and the concentration of cytokinin metabolites were measured. The regulation of gene expression was assayed using transient activation of the promoter of STAY-GREEN2 (SGR2) by cytokinin response regulators. • Key Results While the total amount of cytokinin increased in fruit of both species during maturationand ripening, a high level of expression of two cytokinin biosynthetic gene family members, adenylate isopentenyltransferases, was only detected in green kiwifruit fruit during ripening. Additionally, high levels of O-glucosylated cytokinins were detected only in green kiwifruit, as was the expression of the gene for zeatin O-glucosyltransferase, the enzyme responsible for glucosylating cytokinin into a storage form. Season to season variation in gene expression was seen, and some de-greening of the green kiwifruit fruit occurred in the second season, suggesting environmental effects on the chlorophyll degradation pathway. Two cytokinin-related response regulators, RRA17 and RRB120, showed activity against the promoter of kiwifruit SGR2. • Conclusions The results show that in kiwifruit, levels of cytokinin increase markedly during fruit ripening, and that cytokinin metabolism is differentially regulated in the fruit of the green and gold species. However, the causal factor(s) associated with the maintenance or loss of chlorophyll in kiwifruit during ripening remains obscure.
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