EMS-induced mutagenesis in Choy sum (Brassica chinensis var. parachinensis) and selection for low light tolerance using abiotic stress indices.

BACKGROUND: Choy Sum (Brassica rapa ssp. chinensis var. parachinensis), grown in a controlled environment, is vulnerable to changes in indoor light quality and displays distinct photo-morphogenesis responses. The scarcity of Choy Sum germplasm for indoor cultivation necessitates the development of new cultivars. Hence, this study attempted to develop mutants through chemical mutagenesis and select low-light-tolerant mutants by using abiotic stress tolerance indices. RESULTS: A mutant population of Choy Sum created using 1.5% ethyl methane sulfonate (EMS) at 4 h was manually pollinated to obtain the M2 generation. 154 mutants with reduced hypocotyl length were initially isolated from 3600 M2 seedlings screened under low light (R: FR = 0.5). Five mutants that showed reduced plant height at mature stages were selected and screened directly for shade tolerance in the M3 generation. Principal component analysis based on phenotypic data distinguished the M3 mutants from the wild type. Abiotic stress tolerance indices such as relative stress index (RSI), stress tolerance index (STI), geometric mean productivity (GMP), yield stability index (YSI), and stress resistance index (SRI) showed significant (P < 0.05), and positive associations with leaf yield under shade. M3-12-2 was selected as a shade-tolerant mutant based on high values of STI, YSI, and SRI with low values for tolerance (TOL) and stress susceptibility index (SSI). CONCLUSIONS: The results demonstrate that mutation breeding can be used to create dominant mutants in Choy Sum. Furthermore, we show that screening for low light and selection based on abiotic tolerance indices allowed the identification of mutants with high resilience under shade. This method should apply to developing new cultivars in other crop plants that can be suitable for controlled environments with stable yield performance.

HISTONE DEACETYLASE 9 promotes hypocotyl-specific auxin response under shade.

Vegetative shade causes an array of morphological changes in plants called shade avoidance syndrome, which includes hypocotyl and petiole elongation, leaf hyponasty, reduced leaf growth, early flowering and rapid senescence. Here, we show that loss-of-function mutations in HISTONE DEACETYLASE 9 (HDA9) attenuated the shade-induced hypocotyl elongation in Arabidopsis. However, the hda9 cotyledons and petioles under shade were not significantly different from those in wild-type, suggesting a specific function of HDA9 in hypocotyl elongation in response to shade. HDA9 expression levels were stable under shade and its protein was ubiquitously detected in cotyledon, hypocotyl and root. Organ-specific transcriptome analysis unraveled that shade induced a set of auxin-responsive genes, such as SMALL AUXIN UPREGULATED RNAs (SAURs) and AUXIN/INDOLE-3-ACETIC ACIDs (AUX/IAAs) and their induction was impaired in hda9-1 hypocotyls. In addition, HDA9 binding to loci of SAUR15/65, IAA5/6/19 and ACS4 was increased under shade. The genetic and organ-specific gene expression analyses further revealed that HDA9 may cooperate with PHYTOCHROME-INTERACTING FACTOR 4/7 in the regulation of shade-induced hypocotyl elongation. Furthermore, HDA9 and PIF7 proteins were found to interact together and thus it is suggested that PIF7 may recruit HDA9 to regulate the shade/auxin responsive genes in response to shade. Overall, our study unravels that HDA9 can work as one component of a hypocotyl-specific transcriptional regulatory machinery that activates the auxin response at the hypocotyl leading to the elongation of this organ under shade.

LONG HYPOCOTYL IN FAR-RED 1 mediates a trade-off between growth and defence under shade in Arabidopsis.

Plants respond to vegetative shade with developmental and physiological changes that are collectively known as shade avoidance syndrome (SAS). Although LONG HYPOCOTYL IN FAR-RED 1 (HFR1) is known to be a negative regulator of SAS by forming heterodimers with other basic helix-loop-helix (bHLH) transcription factors to inhibit them, its function in genome-wide transcriptional regulation has not been fully elucidated. Here, we performed RNA-sequencing analyses of Arabidopsis thaliana hfr1-5 mutant and HFR1 overexpression line [HFR1(ΔN)-OE] to comprehensively identify HFR1-regulated genes at different time points of shade treatment. We found that HFR1 mediates the trade-off between shade-induced growth and shade-repressed defence, by regulating the expression of relevant genes in the shade. Genes involved in promoting growth, such as auxin biosynthesis, transport, signalling and response were induced by shade but suppressed by HFR1 under both short and long durations of shade. Likewise, most ethylene-related genes were shade-induced and HFR1-repressed. However, shade suppressed defence-related genes, while HFR1 induced their expression, especially under long durations of shade treatment. We demonstrated that HFR1 confers increased resistance to bacterial infection under shade.

Comparative phenotypic and transcriptomic analyses unravel conserved and distinct mechanisms underlying shade avoidance syndrome in Brassicaceae vegetables.

BACKGROUND: Plants grown under shade are exposed to low red/far-red ratio, thereby triggering an array of altered phenotypes called shade avoidance syndrome (SAS). Shade negatively influences plant growth, leading to a reduction in agricultural productivity. Understanding of SAS is crucial for sustainable agricultural practices, especially for high-density indoor farming. Brassicaceae vegetables are widely consumed around the world and are commonly cultivated in indoor farms. However, our understanding of SAS in Brassicaceae vegetables and their genome-wide transcriptional regulatory networks are still largely unexplored. RESULTS: Shade induced common signs of SAS, including hypocotyl elongation and reduced carotenoids/anthocyanins biosynthesis, in two different Brassicaceae species: Brassica rapa (Choy Sum and Pak Choy) and Brassica oleracea (Kai Lan). Phenotype-assisted transcriptome analysis identified a set of genes induced by shade in these species, many of which were related to auxin biosynthesis and signaling [e.g. YUCCA8 (YUC8), YUC9, and INDOLE-3-ACETIC ACID INDUCIBLE (IAAs)] and other phytohormones signaling pathways including brassinosteroids and ethylene. The genes functioning in plant defense (e.g. MYB29 and JASMONATE-ZIM-DOMAIN PROTEIN 9) as well as in biosynthesis of anthocyanins and glucosinolates were repressed upon shade. Besides, each species also exhibited distinct SAS phenotypes. Shade strongly reduced primary roots and elongated petioles of B. oleracea, Kai Lan. However, these SAS phenotypes were not clearly recognized in B. rapa, Choy Sum and Pak Choy. Some auxin signaling genes (e.g. AUXIN RESPONSE FACTOR 19, IAA10, and IAA20) were specifically induced in B. oleracea, while homologs in B. rapa were not up-regulated under shade. Contrastingly, shade-exposed B. rapa vegetables triggered the ethylene signaling pathway earlier than B. oleracea, Kai Lan. Interestingly, shade induced the transcript levels of LONG HYPOCOTYL IN FAR-RED 1 (HFR1) homolog in only Pak Choy as B. rapa. As HFR1 is a key negative regulator of SAS in Arabidopsis, our finding suggests that Pak Choy HFR1 homolog may also function in conferring higher shade tolerance in this variety. CONCLUSIONS: Our study shows that two Brassicaceae species not only share a conserved SAS mechanism but also exhibit distinct responses to shade, which will provide comprehensive information to develop new shade-tolerant cultivars that are suitable for high-density indoor farms.

Combination of red and blue light induces anthocyanin and other secondary metabolite biosynthesis pathways in an age-dependent manner in Batavia lettuce.

Lettuce is commonly consumed around the world, spurring the cultivation of green- and red-leaf varieties in indoor farms. One common consideration for indoor cultivation is the light wavelengths/spectrum, which is an important factor for regulating growth, development, and the accumulation of metabolites. Here, we show that Batavia lettuce (Lactuca sativa cv. "Batavia") grown under a combination of red (R) and blue (B) light (RB, R:B = 3:1) displayed better growth and accumulated more anthocyanin than lettuce grown under fluorescent light (FL). Anthocyanin concentration was also higher in mature stage than early stage lettuce. By performing a comparative transcriptome analysis of early and mature stage lettuce grown under RB or FL (RB or FL-lettuce), we found that RB induced the expression of genes related to oxidation-reduction reaction and secondary metabolite biosynthesis. Furthermore, plant age affected the transcriptome response to RB, as mature RB-lettuce had six times more differentially expressed genes than early RB-lettuce. Also, genes related to the accumulation of secondary metabolites such as flavonoids and anthocyanins were more induced in mature RB-lettuce. A detailed analysis of the anthocyanin biosynthesis pathway revealed key genes that were up-regulated in mature RB-lettuce. Concurrently, branching pathways for flavonol and lignin precursors were down-regulated.

Rapid metabolite response in leaf blade and petiole as a marker for shade avoidance syndrome.

BACKGROUND: Shade avoidance syndrome (SAS) commonly occurs in plants experiencing vegetative shade, causing morphological and physiological changes that are detrimental to plant health and consequently crop yield. As the effects of SAS on plants are irreversible, early detection of SAS in plants is critical for sustainable agriculture. However, conventional methods to assess SAS are restricted to observing for morphological changes and checking the expression of shade-induced genes after homogenization of plant tissues, which makes it difficult to detect SAS early. RESULTS: Using the model plant Arabidopsis thaliana, we introduced the use of Raman spectroscopy to measure shade-induced changes of metabolites in vivo. Raman spectroscopy detected a decrease in carotenoid contents in leaf blades and petioles of plants with SAS, which were induced by low Red:Far-red light ratio or high density conditions. Moreover, by measuring the carotenoid Raman peaks, we were able to show that the reduction in carotenoid content under shade was mediated by phytochrome signaling. Carotenoid Raman peaks showed more remarkable response to SAS in petioles than leaf blades of plants, which greatly corresponded to their morphological response under shade or high plant density. Most importantly, carotenoid content decreased shortly after shade induction but before the occurrence of visible morphological changes. We demonstrated this finding to be similar in other plant species. Comprehensive testing of Brassica vegetables showed that carotenoid content decreased during SAS, in both shade and high density conditions. Likewise, carotenoid content responded quickly to shade, in a manner similar to Arabidopsis plants. CONCLUSIONS: In various plant species tested in this study, quantification of carotenoid Raman peaks correlate to the severity of SAS. Moreover, short-term exposure to shade can induce the carotenoid Raman peaks to decrease. These findings highlight the carotenoid Raman peaks as a biomarker for early diagnosis of SAS in plants.