White cotton is the dominant natural fibre, accounting for a $USD 36 billion share of the $USD 1.5 trillion global textile industry and, for many decades, has been dyed post-production. Modern cotton ginning and spinning processes require longer and stronger fibres, favouring superior white cotton varieties, which are more amenable to post-harvest dyeing (Gong et al., 2018; Vreeland, 1999). However, large quantities of synthetic dyes from textile dyeing released into the environment/waterways are harming the health of humans and other organisms (Manzoor and Sharma, 2020). Eco-friendly alternatives are urgently needed to reduce pollution and save water; coloured dye-free cotton could be a solution. While naturally coloured cotton has been known for more than 5000 years and occurs in all four species of cultivated cotton, that is Gossypium (G.) hirsutum, G. barbadense, G. herbaceum and G. arboretum (Murthy, 2001; Vreeland, 1999), these coloured varieties generally have low yield, poor fibre quality and variable and unstable colours (Rathinamoorthy and Parthiban, 2019; Vreeland, 1999). Although conventional breeding has improved the properties of some coloured cotton, quality and yield remain low compared with white cotton and colour range is limited (Murthy, 2001; Rathinamoorthy and Parthiban, 2019). Betalains are tyrosine-derived pigments found naturally in the order Caryophyllales of flowering plants, and fungi and bacteria (Timoneda et al., 2019). Betalains comprise two classes of compounds, that is yellow-orange betaxanthins and red-violet betacyanins. These compounds are synthesized through a series of enzymatic steps including hydroxylases, dioxygenases and glucosyl transferases that produce visible colours. As the Malvaceae typically do not produce betalains, we embarked on genetically engineering the betalain pathway in G. hirsutum, the world's largest plant-based fibre commodity. We designed constructs that included the coding sequences (CDS) of BvDODA1 (Beta vulgaris, GeneBank ID HQ656027.1), BvCYP76AD1 (HQ656023.1) and MjcDOPA5GT (Mirabilis jalapa, AB182643.1; Polturak et al., 2017; Timoneda et al., 2019; Figure 1a,b). The CDSs were optimized for Arabidopsis and synthesized by GeneArt (Table S1). Constructs (pAGM4723 vector) were assembled via Golden Gate cloning with a 2 × 35 S-driven kanamycin resistance gene. The betalain genes were driven by either a 2 × 35 S constitutive promoter or a ltp3/8K12 (LTP) mid-late-stage cotton fibre-specific promoter (Figure 1b, Table S1). Transgenic plants were generated through tissue culture transformation (Murray et al., 1999; Figure 1c). Coker 315-11 was used as the recipient of transformation by infection with Agrobacterium tumefaciens strain AGL1 containing different constructs. Transgenic and wild-type (controls) cotton plants were grown in a greenhouse at 28°C/20°C (day/night) with natural light. The pattern of betalain production differed strongly between these two promoters. Under the fibre-specific promoter, betalain pigments were successfully produced in cotton fibres and resulted in visibly pink fibres (Figure 1d–f). Plants were either green or partially purple (leaky expression in old tissues) and flowers resembled those of control plants at 0 days postanthesis (DPA; Figure S1). By contrast, the constitutive promoter-driven construct generated purple/violet plants with purple leaves, stems, boll coats and bracts (Figure S1). The flowers were pink at opening (0 DPA) while wild type had green leaves with creamy yellow flowers (Figure S1). However, constitutive promoter-driven constructs generated white fibres throughout development, similar to wild type, although ovules and mature seed interior were pink/purple (Figure S1). Despite strong betalain accumulation during fibre development in the fibre-specific lines, the colour faded to light brown/pink in the final days of boll maturation, when the bolls dried and opened (transgenic lines: 55–60 DPA vs wild type: 55 DPA; Figures 1e, S1). This suggests that vacuole-located betalain (Figure S2) was degraded during the final maturation stage. Cotton bolls were collected at around 10, 15, 20, 25, 30, 40, 50 and 60 DPA, and boll coats were either cut open or removed entirely followed by 48 h of freeze-drying, and the pink colour was retained (Figures 1f, S1). Mature fibre or freeze-dried immature fibre (>46 DPA) from controls and five independent T0 plants with the fibre-specific betalain expression were measured by Cottonscope for fibre quality. The results suggested the transgenic lines have the potential to present similar maturity ratio and fineness as the wild type (Line 44, Figure 1g). The seed numbers and fibre yield were less in the T0 compared with wild type, which is common to see in T0 generation (Figure S3). Subsequent generations of transgenic plants could potentially retain wild-type-like yield and fibre quality alongside strong pigment accumulation, noting that Coker 315 can be introgressed into modern elite varieties for yield and quality. New colours may be generated by crossing the betalain lines with existing naturally coloured genotypes (Ke et al., 2022). In conclusion, we present a novel example of introducing the exogenous multi-gene betalain pathway to generate a plant-made pink cotton fibre which remains pink until the very late stages of fibre development. Using the betalain pathway is advantageous because the pigment is stable over a wide pH range (Jackman and Smith, 1996), potentially improving colour stability and consistency. Future research could investigate reducing pigment degradation (potentially via cross-linking) and introducing new colours. We thank Lissette Perez and Dina Yulia for technical support, Drs Danny Llewellyn and Phil Hands for helpful advice and support, and the CSIRO Synthetic Traits Group for Golden Gate vectors and support. The authors declare no conflict of interest. This research was funded by the CSIRO SynBio Future Science Platform. MM, VR, CM and FP contributed to the conceptualization. MM, VR, CM, FP and XL contributed to the methodology. XL contributed to the formal analysis. SA, MM, XL, VR, CM and FP contributed to the investigation. XL contributed to the writing—original draft. XL, MM, CM, FP and VR contributed to the writing—review and editing. CM, FP and VR contributed to the supervision. FP, MM, CM and XL contributed to the project administration. FP and CM contributed to funding acquisition. The data are available in the article and accompanying supplementary material. Table S1 Betalain-pathway gene coding sequences (CDS) and LTP promoter. Figure S1 Images: cotton plants, seeds, mature bolls, freeze-dried immature bolls, stem sections and T1 plants. Figure S2 Betalain fluorescence. Figure S3 Yield of T0. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

In Australia, digital agriculture is considered immature and its adoption ad hoc, despite a relatively advanced technology innovation sector. In this review, we focus on the technical, governance and social factors of digital adoption that have created a disconnect between technology development and the end user community (farmers and their advisors). Using examples that reflect both successes and barriers in Australian agriculture, we first explore the current enabling technologies and processes, and then we highlight some of the key socio-technical factors that explain why digital agriculture is immature and ad hoc. Pronounced issues include fragmentation of the innovation system (and digital tools), and a lack of enabling legislation and policy to support technology deployment. To overcome such issues and increase adoption, clear value propositions for change are necessary. These value propositions are influenced by the perceptions and aspirations of individuals, the delivery of digitally-enabled processes and the supporting legislative, policy and educational structures, better use/conversion of data generated through technology applications to knowledge for supporting decision making, and the suitability of the technology. Agronomists and early adopter farmers will play a significant role in closing the technology-end user gap, and will need support and training from technology service providers, government bodies and peer-networks. Ultimately, practice change will only be achieved through mutual understanding, ownership and trust. This will occur when farmers and their advisors are an integral part of the entire digital innovation system.

Summary Engineering high biomass plants that produce oil (triacylglycerol or TAG) in vegetative rather than seed‐related tissues could help meet our growing demand for plant oil. Several studies have already demonstrated the potential of this approach by creating transgenic crop and model plants that accumulate TAG in their leaves and stems. However, TAG synthesis may compete with other important carbon and energy reserves, including carbohydrate production, and thereby limit plant growth. The aims of this study were thus: first, to investigate the effect of TAG accumulation on growth and development of previously generated high leaf oil tobacco plants; and second, to increase plant growth and/or oil yields by further altering carbon fixation and partitioning. This study showed that TAG accumulation varied with leaf and plant developmental stage, affected leaf carbon and nitrogen partitioning and reduced the relative growth rate and final biomass of high leaf oil plants. To overcome these growth limitations, four genes related to carbon fixation (encoding CBB cycle enzymes SBPase and chloroplast‐targeted FBPase) or carbon partitioning (encoding sucrose biosynthetic enzyme cytosolic FBPase and lipid‐related transcription factor DOF4) were overexpressed in high leaf oil plants. In glasshouse conditions, all four constructs increased early growth without affecting TAG accumulation while chloroplast‐targeted FBPase and DOF4 also increased final biomass and oil yields. These results highlight the reliance of plant growth on carbon partitioning, in addition to carbon supply, and will guide future attempts to improve biomass and TAG accumulation in transgenic leaf oil crops.

This article examines contemporary representations of female and male scientists in The New York Times with a particular emphasis on stereotypes related to gender and science as a profession. The selected series of profiles is approximately proportional in its representation of women in science and generally gives a rounded and diverse picture of their subjects. Traditionally ‘masculine’ characteristics (e.g. individual drive and brilliance) as well as ‘feminine’ communal skills (e.g. collaboration, communication and teamwork) are attributed to both male and female scientists. Nevertheless, textual and image analyses reveal that some differences remain in the treatment of male and female subjects, particularly in the unequal focus on combining family and career. This research identifies progress in media representations of scientists in comparison to previous studies. However, there is still room for improvement, especially in the representation of scientists from culturally and linguistically diverse backgrounds.

Synthesis and accumulation of the storage lipid triacylglycerol in vegetative plant tissues has emerged as a promising strategy to meet the world's future need for vegetable oil. Sorghum (Sorghum bicolor) is a particularly attractive target crop given its high biomass, drought resistance and C4 photosynthesis. While oilseed-like triacylglycerol levels have been engineered in the C3 model plant tobacco, progress in C4 monocot crops has been lagging behind. In this study, we report the accumulation of triacylglycerol in sorghum leaf tissues to levels between 3 and 8.4% on a dry weight basis depending on leaf and plant developmental stage. This was achieved by the combined overexpression of genes encoding the Zea mays WRI1 transcription factor, Umbelopsis ramanniana UrDGAT2a acyltransferase and Sesamum indicum Oleosin-L oil body protein. Increased oil content was visible as lipid droplets, primarily in the leaf mesophyll cells. A comparison between a constitutive and mesophyll-specific promoter driving WRI1 expression revealed distinct changes in the overall leaf lipidome as well as transitory starch and soluble sugar levels. Metabolome profiling uncovered changes in the abundance of various amino acids and dicarboxylic acids. The results presented here are a first step forward towards the development of sorghum as a dedicated biomass oil crop and provide a basis for further combinatorial metabolic engineering.

Carbon-concentrating mechanisms (CCMs) enable efficient photosynthesis and growth in CO2-limiting environments, and in eukaryotic microalgae localisation of Rubisco to a microcompartment called the pyrenoid is key. In the model green alga Chlamydomonas reinhardtii, Rubisco preferentially relocalises to the pyrenoid during CCM induction and pyrenoid-less mutants lack a functioning CCM and grow very poorly at low CO2. The aim of this study was to investigate the CO2 response of pyrenoid-positive (pyr+) and pyrenoid-negative (pyr-) mutant strains to determine the effect of pyrenoid absence on CCM induction and gene expression. Shotgun proteomic analysis of low-CO2-adapted strains showed reduced accumulation of some CCM-related proteins, suggesting that pyr-has limited capacity to respond to low-CO2 conditions. Comparisons between gene transcription and protein expression revealed potential regulatory interactions, since Rubisco protein linker (EPYC1) protein did not accumulate in pyr-despite increased transcription, while elements of the LCIB/LCIC complex were also differentially expressed. Furthermore, pyr-showed altered abundance of a number of proteins involved in primary metabolism, perhaps due to the failure to adapt to low CO2.Carbon- This work highlights two-way regulation between CCM induction and pyrenoid formation, and provides novel candidates for future studies of pyrenoid assembly and CCM function.

Plant storage compounds such as starch and lipids are important for human and animal nutrition as well as industry. There is interest in diverting some of the carbon stored in starch-rich organs (leaves, tubers, and cereal grains) into lipids in order to improve the energy density or nutritional properties of crops as well as providing new sources of feedstocks for food and manufacturing. Previously, we generated transgenic potato plants that accumulate up to 3.3% triacylglycerol (TAG) by dry weight in the tubers, which also led to changes in starch content, starch granule morphology and soluble sugar content. The aim of this study was to investigate how TAG accumulation affects the nutritional and processing properties of high oil potatoes with a particular focus on starch structure, physical and chemical properties. Overall, TAG accumulation was correlated with increased energy density, total nitrogen, amino acids, organic acids and inorganic phosphate, which could be of potential nutritional benefit. However, TAG accumulation had negative effects on starch quality as well as quantity. Starch from high oil potatoes had lower amylose and phosphate content, reduced peak viscosity and higher gelatinization temperature. Interestingly, starch pasting properties were disproportionately affected in lines accumulating the highest levels of TAG (>2.5%) compared to those accumulating only moderate levels (0.2-1.6%). These results indicate that optimized engineering of specialized crops for food, feed, fuel and chemical industries requires careful selection of traits, and an appropriate level of transgene expression, as well as a better understanding of starch structure and carbon partitioning in plant storage organs.

Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.

Synthesis and accumulation of plant oils in the entire vegetative biomass offers the potential to deliver yields surpassing those of oilseed crops. However, current levels still fall well short of those typically found in oilseeds. Here we show how transcriptome and biochemical analyses pointed to a futile cycle in a previously established Nicotiana tabacum line, accumulating up to 15% (dry weight) of the storage lipid triacylglycerol in leaf tissue. To overcome this metabolic bottleneck, we either silenced the SDP1 lipase or overexpressed the Arabidopsis thaliana LEC2 transcription factor in this transgenic background. Both strategies independently resulted in the accumulation of 30-33% triacylglycerol in leaf tissues. Our results demonstrate that the combined optimization of de novo fatty acid biosynthesis, storage lipid assembly and lipid turnover in leaf tissue results in a major overhaul of the plant central carbon allocation and lipid metabolism. The resulting further step changes in oil accumulation in the entire plant biomass offers the possibility of delivering yields that outperform current oilseed crops.

SummaryPotato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber‐specific (patatin) and constitutive (CaMV‐35S) promoters. This coordinated metabolic engineering approach resulted in over a 100‐fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild‐type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof‐of‐concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.

In the model green alga Chlamydomonas reinhardtii, a carbon-concentrating mechanism (CCM) is induced under low CO2 in the light and comprises active inorganic carbon transport components, carbonic anhydrases, and aggregation of Rubisco in the chloroplast pyrenoid. Previous studies have focused predominantly on asynchronous cultures of cells grown under low versus high CO2. Here, we have investigated the dynamics of CCM activation in synchronized cells grown in dark/light cycles compared with induction under low CO2. The specific focus was to undertake detailed time course experiments comparing physiology and gene expression during the dark-to-light transition. First, the CCM could be fully induced 1 h before dawn, as measured by the photosynthetic affinity for inorganic carbon. This occurred in advance of maximum gene transcription and protein accumulation and contrasted with the coordinated induction observed under low CO2. Between 2 and 1 h before dawn, the proportion of Rubisco and the thylakoid lumen carbonic anhydrase in the pyrenoid rose substantially, coincident with increased CCM activity. Thus, other mechanisms are likely to activate the CCM before dawn, independent of gene transcription of known CCM components. Furthermore, this study highlights the value of using synchronized cells during the dark-to-light transition as an alternative means of investigating CCM induction.

Pharmaceutical 1,8-cineole oil is commercially harvested from Eucalyptus polybractea R.T. Baker grown in short rotation coppice cultivation. Harvests occur throughout the year, with no consideration given to the proportion of leaves present at different ontogenetic stages, and the effect leaf ontogeny may have on harvested oil quality and quantity. To determine the optimal leaf stage to harvest pharmaceutical-grade oil, we quantified the relationship between leaf ontogeny and foliar oil quantity and quality, secretory cavity size, and abundance along branches of two E. polybractea clones. The results show that the desirable attributes of oil yield and 1,8-cineole proportion both reach their maximum in fully expanded leaves and that this coincides with maximum secretory cavity size. Across all leaf ages the amount of various monoterpenes were highly correlated with 1,8-cineole, suggesting a single, multi-product monoterpene synthase may be responsible. In the very young leaves, sesquiterpenes such as β-elemene were the most abundant constituents of the essential oil. This study suggests that there may be differential transcriptional regulation of mono- and sesquiterpene synthases with leaf ontogeny such that sesquiterpene biosynthesis is at a maximum only in young leaves, and that monoterpene biosynthesis dominates during leaf and concomitant cavity expansion. Given E. polybractea leaves are produced during major seasonal flushes, there are times throughout the year when the numbers of young leaves with inferior oil quantity and quality are likely to be proportionally decreased. Therefore harvesting times could be manipulated to take advantage of such times and ensure abundant high quality oil is harvested.

The pyrenoid is a subcellular microcompartment in which algae sequester the primary carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The pyrenoid is associated with a CO(2)-concentrating mechanism (CCM), which improves the operating efficiency of carbon assimilation and overcomes diffusive limitations in aquatic photosynthesis. Using the model alga Chlamydomonas reinhardtii, we show that pyrenoid formation, Rubisco aggregation, and CCM activity relate to discrete regions of the Rubisco small subunit (SSU). Specifically, pyrenoid occurrence was shown to be conditioned by the amino acid composition of two surface-exposed α-helices of the SSU: higher plant-like helices knock out the pyrenoid, whereas native algal helices establish a pyrenoid. We have also established that pyrenoid integrity was essential for the operation of an active CCM. With the algal CCM being functionally analogous to the terrestrial C(4) pathway in higher plants, such insights may offer a route toward transforming algal and higher plant productivity for the future.

BACKGROUND:The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils.RESULTS:Leaves were digested using a variety of commercially available enzymes. A pectinase from Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h), with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories.CONCLUSIONS:The protocol described herein is likely to be adaptable to a range of Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain.