Zhiguo Liu

SUMMARY: Photosynthesis impairment and the accompanying leaf yellowing represent a prominent and physiologically critical symptom of phytoplasma infection. However, the underlying molecular mechanisms remain poorly understood. In this study, we identified the Jujube Witches' Broom pathogenic effector PHYL1 _JWB , which was previously reported to induce phyllody in jujube, as a key inducer of photosynthesis impairment. Overexpression of PHYL1 _JWB in sour jujube and Arabidopsis thaliana disrupted photosynthesis by causing severe disorganization of chloroplast ultrastructure. PHYL1 _JWB directly interacts with the integral thylakoid membrane protein ZjCURT1A and promotes its 26S proteasome‐dependent degradation independently of lysine ubiquitination, leading to disruption of thylakoid structure and photosynthetic dysfunction. The conserved two α‐helices of PHYL1 _JWB and the C‐terminal segment of ZjCURT1A containing the thylakoid membrane curvature‐related CAAD motif are essential for their interaction. Together, these findings reveal that PHYL1 _JWB impairs photosynthesis by disrupting chloroplast ultrastructural in addition to inducing phyllody, thereby contributing to abnormal plant growth. This work highlights the functional pleiotropy of pathogenic effectors and underscores the need to consider their impacts on multiple physiological processes in the context of phytoplasma pathogenicity.

BBX transcription factors are key regulators of plant growth, development, and stress responses, yet their functions in jujube (Ziziphus jujuba Mill.), an economically important fruit tree, remain largely unexplored. Here, we systematically identified 26 ZjBBX genes and comprehensively analyzed their phylogenetic relationships, structural features, and expression dynamics. Our integrative approach revealed three major findings. First, nine ZjBBX genes exhibited distinct expression patterns during fruit coloration, with ZjBBX9 and ZjBBX25 emerging as persistent up-regulated candidates. Second, under salt stress, ZjBBX members displayed three contrasting response modes, sustained down-regulation, rapid induction, or sharp repression, suggesting functional diversification in salinity adaptation. Third, under jujube witches’ broom, ZjBBX8, ZjBBX11, and ZjBBX13 showed differential expression between healthy and infected materials, implicating them in biotic stress defense. Collectively, this study provides the first multidimensional expression atlas of the ZjBBX family linked to fruit quality, salt tolerance, and pathogen resistance in jujube. Our findings open new avenues for marker-assisted breeding and genetic improvement of stress-resilient jujube cultivars.

SUMMARY: Ziziphus jujuba Mill. ‘Dongzao’ (‘Dongzao’) is the most widely planted fresh jujube cultivar in China. Ziziphus jujuba Mill. ‘Riguang’ (‘Riguang’), obtained through colchicine‐induced mutagenesis, exhibits superior fruit size and quality compared to ‘Dongzao’, but its significantly reduced cold tolerance limits large‐scale cultivation. To elucidate the molecular mechanisms underlying the differential cold tolerance between ‘Dongzao’ and ‘Riguang’, we examined the expression levels of ZjCML41 , ZjCBF1 , ZjCBF3 , and ZjICE2 , which were significantly upregulated in ‘Dongzao’ relative to ‘Riguang’. Overexpression of ZjCML41 in Arabidopsis and jujube increased cold tolerance, whereas transient silencing of ZjCML41 in jujube decreased cold tolerance. Additionally, ZjbZIP25 could interact with the promoter region of ZjCML41 and positively regulate its expression, thereby enhancing cold tolerance. Moreover, ZjCML41 interacted with ZjIQD15, which in turn positively influenced cold tolerance by modulating the ICE‐CBF pathway and reactive oxygen species scavenging activity. The proposed regulatory module comprising ZjbZIP25 , ZjCML41 , and ZjIQD15 enhances cold tolerance in ‘Dongzao’ by activating the ICE‐CBF pathway, while downregulation of these genes reduces cold tolerance in ‘Riguang’. These findings provide valuable theoretical insights and potential practical strategies for improving and regulating cold tolerance in ‘Riguang’.

Pectin methylesterases (PMEs) are essential enzymes that catalyze the demethylesterification of pectins, thereby facilitating their degradation. They have been implicated in fruit ripening, but their role in fruit size regulation remains unclear. This study found that ZjPME36 was more highly expressed in small-fruit cultivars than in large-fruit cultivars of Chinese jujube (Ziziphus jujuba Mill.). Overexpression of ZjPME36 in tomato significantly reduced the fruit size by restricting mesocarp cell expansion. Concurrently, the content of PME increased significantly, while both the degree of pectin methylesterification and total pectin content decreased. Furthermore, the transcription factors ZjPosF21 and ZjWRKY48 were identified as an activator and a repressor of ZjPME36, respectively. They coordinately contribute to the final fruit size of jujube by altering pectin content. This research sheds new light on the molecular pathway for PME-mediated fruit size regulation in jujube.

Phytoplasma (‘ Candidatus Phytoplasma’), a bacterial pathogen, is a significant plant health concern worldwide, resulting in substantial economic losses. In jujube ( Ziziphus jujuba ), it is often referred to as a cancer‐like disease that can destroy the whole plant and cause great economic loss, posing serious challenges to jujube's sustainable development. Here, the phytoplasma tolerance mechanism in jujube was revealed by the identification of a highly phytoplasma‐tolerant jujube genotype. Integrated transcriptomic and metabolomic analyses were conducted to compare a phytoplasma‐susceptible genotype Fuxiang with a tolerant genotype T13. The results revealed that the ZjWRKY40 gene was significantly upregulated in T13 about 3‐fold at the third development stage, suggesting its key role in mediating phytoplasma tolerance. A phytoplasma effector SJP4 _JWB was identified and shown to interact with ZjWRKY40 by yeast two‐hybrid and co‐immunoprecipitation methods. In addition, ZjWRKY40 was found to bind to the promoter of zju‐miR157 , thereby regulating its expression. Moreover, zju‐miR157 targeted ZjSPL3 and negatively affected the phytoplasma tolerance by downregulating ZjSPL3 expression. Together, these findings outline a regulatory network involving SJP4 _JWB –ZjWRKY40–zju‐miR157 , which provides important insights into the molecular mechanism of the phytoplasma tolerance in jujube and lays a foundation for developing tolerant genotypes through molecular breeding.

Phytoplasmas can induce complex and substantial phenotypic changes in their hosts in ways that favour their colonisation, but the mechanisms underlying these changes remain largely unknown. Jujube witches' broom (JWB) disease is a typical phytoplasma disease causing great economic loss in Chinese jujube (Ziziphus jujuba Mill.). Here, we reported an effector, PHYL1_JWB from Candidatus Phytoplasma ziziphi, which implicated in inducing abnormal floral organogenesis. Utilising a combination of in vivo and in vitro methods, we investigated the influence of PHYL1_JWB on the proteins associated with floral development. Our findings reveal that PHYL1_JWB facilitates the proteasome‐mediated degradation of essential flower morphogenetic regulators, including AP1, SEP1, SEP2, SEP3, SEP4, CAL, and AGL6, through a distinctive pathway that is dependent on the activity of the 26S proteasome, thus obviating the requirement for lysine ubiquitination of the substrates. Further, the Y2H analysis showed that the leucine at position 75th in second α helix of PHYL1_JWB is fundamental for the interactions of PHYL1_JWB with AP1 and SEP1‐4 in jujube and Arabidopsis. Our research carry profound implications for elucidating the contribution of PHYL1_JWB to the aberrant floral development in diseased jujube, and help to establish a robust theoretical underpinning for the prophylaxis and therapy of JWB disease.

Fruit size is crucial for fruit trees, as it contributes to both quality and yield. However, the underlying mechanism of fruit size regulation remains largely unknown. Taking advantage of using a fruit double-sized bud mutant of Chinese jujube, "Jinkuiwang" and its wild type, "Jinsixiaozao", we carried out a comprehensive study on the mechanism of fruit size development in jujube. Using weighted gene coexpression network analyses, a number of candidate regulators for fruit size including those involved in hormonal signaling pathways, transcription factors, and heat shock proteins were identified. A hub gene named cytokinin oxidase/dehydrogenase 5 (ZjCKX5), responsible for cytokinin degradation, was found to play a negative role in regulating fruit size development, and overexpressing ZjCKX5 in tomato and Arabidopsis resulted in much smaller fruits and dwarf plants. Furthermore, another two hub genes, ZjWRKY23 and ZjWRKY40 transcription factors, were found to participate in fruit size regulation by targeting and downregulating the ZjCKX5 expression. Overexpressing ZjWRKY23 or ZjWRKY40 in tomato led to much larger fruits and promoted plant architecture. Based on these results, a molecular framework for jujube fruit size regulation, namely, ZjWRKY-ZjCKX5 module, was proposed. This study provides a new insight into the molecular networks underlying fruit size regulation.

The Ziziphus mauritiana Lam. and Z. jujuba Mill. are the two most economically important members of the genus Ziziphus. The fruit color of Z. mauritiana remains green throughout fruit development in the majority of commercial cultivars, whereas its close relative, Z. jujuba Mill. turns from green to red in all cultivars. However, the lack of transcriptomic and genomic information confines our understanding of the molecular mechanisms underlying fruit coloration in Z. mauritiana (Ber). In the present study, we performed the transcriptome-wide analysis of MYB transcription factors (TFs) genes in Z. mauritiana and Z. jujuba, and identified 56 ZmMYB and 60 ZjMYB TFs in Z. mauritiana and Z. jujuba, respectively. Through transcriptomic expression analysis, four similar MYB genes (ZmMYB/ZjMYB13, ZmMYB/ZjMYB44, ZmMYB/ZjMYB50, and ZmMYB/ZjMYB56) from Z. mauritiana and Z. jujuba were selected as candidate key genes regulating flavonoid biosynthesis. Among these genes, the ZjMYB44 gene was transiently highly expressed in fruit, and flavonoid content accumulation also increased, indicating that this gene can influence flavonoid content during the period of fruit coloration in Z. jujuba. The current study adds to our understanding of the classification of genes, motif structure, and predicted functions of the MYB TFs, as well as identifying MYBs that regulate flavonoid biosynthesis in Ziziphus (Z. mauritiana and Z. jujuba). Based on this information, we concluded that MYB44 is involved in the flavonoids biosynthesis pathway during the fruit coloring of Ziziphus. Our research results provide an important understanding of the molecular mechanism of flavonoid biosynthesis resulting in fruit coloration and laying a foundation for further genetic improvement of fruit color in Ziziphus.

Adenylate cyclase is the key enzyme solely synthesizing cAMP which participates in cell metabolism regulations and functions as an intracellular second messenger. However, the biological functions of plant ACs have not been elucidated clearly for their poor conservative sequences and low detectable cAMP. We performed a systematic study of plant ACs by using Chinese jujube, whose fruit exhibits the highest cAMP content among plants. Three novel ACs were identified from Chinese jujube, and two types of methods including in vitro and in vivo were used to certificate ZjAC1-3 which can catalyze the conversion of ATP into cAMP. The biological functions of significant accelerations of seed germination, root growth, and flowering were found via overexpression of these AC genes in Arabidopsis, and these functions of ACs were further demonstrated by treating the AC-overexpressing transgenic lines and wild type Arabidopsis with bithionol and dibutyryl-cAMP. At last, transcriptome data revealed that the underlying mechanism of the biological functions of ACs might be regulation of the key genes involved in the circadian rhythm pathway and the hormone signal transduction pathway. This research established a foundation for further investigating plant AC genes and provided strong evidence for cAMP serving as a signaling molecule in plants.