A Heat Stress Responsive NAC Transcription Factor Heterodimer Plays Key Roles in Rice Grain Filling

Abstract

22High temperature usually leads to the failure of grain filling during caryopsis 23 development, resulting in the loss of yield, however the mechanism is not yet well 24 elucidated. Here, we report that two rice caryopsis-specific NAM/ATAF/CUC domain 25 transcription factors, ONAC127 and ONAC129, respond to heat stress and are involved 26 in the caryopsis filling process. ONAC127 and ONAC129 are dominantly expressed in 27 pericarp during grain filling and can form a heterodimer. To investigate the functions 28 of these two ONACs, we obtained CRISPR/cas9 induced mutants and overexpression 29 lines of them. Interestingly, we found that both knock-out and overexpression plants 30showed incompletely filling and shrunken phenotype of caryopses, which became more 31 severe under heat stress. The shrunken caryopses of these transgenic lines are usually 32 with ectopic accumulation of starch in the pericarp. Transcriptome analyses revealed 33 that ONAC127 and ONAC129 mainly regulate stimulus response, cell wall construction 34 and nutrient transport etc. ChIP-seq analyses identified the direct targets of ONAC127 35 and ONAC129 in developing caryopses, including monosaccharide transporter 36OsMST6, sugar transporter OsSWEET4, calmodulin-like protein OsMSR2 and 37Ethylene-Response AP2/ERF Factor OsEATB. The result suggested that ONAC127 and 38 ONAC129 might regulate the caryopsis filling through sugar transportation and abiotic 39 stress responses. Overall, this study demonstrates the transcriptional regulatory 40 networks involving ONAC127 and ONAC129, which coordinates multiple pathways 41 to modulate caryopsis development and heat stress response at rice filling stage. 42 43 rice caryopsis is mainly composed of endosperm filled with starch grains, and the 48 biosynthesis of starch is closely related to carbohydrates transportation. Carbohydrates 49 are synthesized in leaves and delivered to caryopses via phloem (Patrick, 1997), then 50 unloaded to the pericarp as the energy and materials for starch biosynthesis (Zhang et 51 al., 2007). The dorsal vascular bundle passes through the pericarp is the main nutrient 52 transport tissue in caryopsis (Oparka and Gates, 1981). While the vascular bundles are 53 not contiguous with endosperm tissue (Hoshikawa, 1984), the apoplasmic pathway is 54 the only way for nutrient to reach the starchy endosperm. (Matsuda et al., 1979). 55Carbohydrates may enter the nucellar epidermis directly through plasmodesmata, 56 then be transported to the apoplasmic space with sugar transporter protein OsSWEETs 57 (sugar will eventually be exported transporters) and partially hydrolyzed into 58 monosaccharide by cell wall invertase OsCINs. The monosaccharide is transported into 59 the aleurone layer mainly by monosaccharide transporter OsMSTs while the rest un-60 hydrolyzed sucrose is directly transported into the aleurone layer via sucrose transporter 61OsSUTs (Yang et al., 2018). The nutrient transportation is also regulated by a range of 62 transcription factors such as OsNF-YB1 and OsNF-YC12, ...

OsAT1, an anion transporter, negatively regulates grain size and yield in rice

“…Furthermore, the Golgi marker (G‐968) and endoplasmic reticulum marker (ER‐960) were co‐transformed into rice protoplasts according to the method described in Nelson et al (2007). The fluorescent signals were detected using an SP8 confocal laser scanning microscope (TCS, Leica, German, https://www.leicabiosystems.com/) using the channel specifications described in Ren et al (2021).…”

Time to retire? A life‐changing decision made by NAC transcription factors

“…NAC transcription factors are active as homodimers or heterodimers with other NACs. Rice ONAC127 is able to activate expression when assayed on its own; however, when assayed alongside its heterodimer partner ONAC129, its activity is repressed (Ren et al ., 2021). ONAC129 on its own is also an activator, so this cannot be simply explained as ONAC129 repressing ONAC127.…”

Meta-QTLs, ortho-MQTLs and candidate genes for thermotolerance in wheat (Triticum aestivum L.)

“…The crucial roles of some of these genes associated with Ortho-MQTLs in conferring thermotolerance can be summarized as follows: (i) mitogen-activated protein kinase cascades have been shown to be involved in signalling pathways activated by different abiotic stresses including heat stress in different plant species (Sinha et al 2011), (ii) polyamine transporter is required for the uptake of extracellular polyamines and is known to play a key role in stabilizing the mRNAs of several important heat stress-responsive genes under heat stress in Arabidopsis (Shen et al 2016), (iii) CRISPR/Cas9 based genome editing and transgenesis demonstrated a transcriptional regulatory network with two NAC genes (ONAC127 and ONAC129) coordinating multiple pathways to modulate grain development and heat stress responses at reproductive stages in rice (Ren et al 2021), (iv) a mitochondrial nucleoside diphosphate kinase (mtNDPK) is known to be involved in heat stress response in pea, possibly as a modulator of a protein (86 kilo-dalton protein) synthesized upon exposure to heat (Escobar et al 2001), (v) phospholipase D and phosphatidylinositol 4,5-bisphosphate are accumulated at the plasma membrane and nucleus, thereby regulates the ion channels and the cytoskeleton under heat stress in Arabidopsis and rice (Mishkind et al 2009), and (vi) the RNA recognition motif of Arabidopsis splicing factor affects the alternative splicing of pre-mRNAs of genes, including FLOWERING LOCUS M and a heat shock transcription factor involved in the regulation of owering and adaptation to heat stress (Lee et al 2017). Among the remaining ve ortho-MQTLs, only one ortholog matched between wheat and rice MQTLs (Table 4).…”

“…Ortho-MQTL2B.3 contained at least two heat-responsive genes encoding for protein kinase (Sinha et al 2011) and amino acid/polyamine transporter (Shen et al 2016), respectively. Whereas, ortho-MQTL2D.1 included at least 5 heat-responsive genes encoding for the following proteins/products: NAC domain-containing protein (Ren et al 2021), nucleoside phosphatase (Escobar et al 2001), PLD-regulated protein1-like (Mishkind et al 2009), RNA recognition motif domain (Lee et al 2017), and helicase (Gong et al 2005).…”